Biochemical analysis of the NAD+-dependent malate dehydrogenase, a substrate of several serine/threonine protein kinases of Mycobacterium tuberculosis.

PknD is one of the eleven eukaryotic-like serine/threonine protein kinases (STPKs) of Mycobacterium tuberculosis (Mtb). In vitro phosphorylation assays with the active recombinant PknD showed that the intracellular protein NAD+-dependent malate dehydrogenase (MDH) is a substrate of this kinase. MDH, an energy-supplying enzyme, catalyzes the interconversion of malate and oxaloacetate and plays crucial roles in several metabolic pathways including the citric acid cycle. The phosphorylation site was identified on threonine residues and the phosphorylation inhibited the MDH activity. In vitro, the recombinant MDH could also be phosphorylated by at least five other STPKs, PknA, PknE, PknH, PknJ, and PknG. Immunoprecipitation analysis revealed that MDH was hyperphosphorylated in the bacteria at the beginning of the stationary and under oxygen-limited conditions by STPKs other than PknD. On the contrary, when PknD-deficient mutant mycobacteria were grown in a phosphate-depleted medium, MDH was not detectably phosphorylated. These results suggest that although the MDH is a substrate of several mycobacterial STPKs, the activity of these kinases can depend on the environment, as we identified PknD as a key element in the MDH phosphorylation assay under phosphate-poor conditions.

Protective effect of different anti-rabies virus VHH constructs against rabies disease in mice.

Rabies virus causes lethal brain infection in about 61000 people per year. Each year, tens of thousands of people receive anti-rabies prophylaxis with plasma-derived immunoglobulins and vaccine soon after exposure. Anti-rabies immunoglobulins are however expensive and have limited availability. VHH are the smallest antigen-binding functional fragments of camelid heavy chain antibodies, also called Nanobodies. The therapeutic potential of anti-rabies VHH was examined in a mouse model using intranasal challenge with a lethal dose of rabies virus. Anti-rabies VHH were administered directly into the brain or systemically, by intraperitoneal injection, 24 hours after virus challenge. Anti-rabies VHH were able to significantly prolong survival or even completely rescue mice from disease. The therapeutic effect depended on the dose, affinity and brain and plasma half-life of the VHH construct. Increasing the affinity by combining two VHH with a glycine-serine linker into bivalent or biparatopic constructs, increased the neutralizing potency to the picomolar range. Upon direct intracerebral administration, a dose as low as 33 µg of the biparatopic Rab-E8/H7 was still able to establish an anti-rabies effect. The effect of systemic treatment was significantly improved by increasing the half-life of Rab-E8/H7 through linkage with a third VHH targeted against albumin. Intraperitoneal treatment with 1.5 mg (2505 IU, 1 ml) of anti-albumin Rab-E8/H7 prolonged the median survival time from 9 to 15 days and completely rescued 43% of mice. For comparison, intraperitoneal treatment with the highest available dose of human anti-rabies immunoglobulins (65 mg, 111 IU, 1 ml) only prolonged survival by 2 days, without rescue. Overall, the therapeutic benefit seemed well correlated with the time of brain exposure and the plasma half-life of the used VHH construct. These results, together with the ease-of-production and superior thermal stability, render anti-rabies VHH into valuable candidates for development of alternative post exposure treatment drugs against rabies.

A two-step lyssavirus real-time polymerase chain reaction using degenerate primers with superior sensitivity to the fluorescent antigen test.

A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤ 1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible.

Infectivity of rabies virus-exposed macrophages.

Rabies virus distributes widely in infected mice, including lymphoid tissues and spleen macrophages. The infection characteristics in murine macrophages and the infectivity of virus-exposed macrophages were examined upon inoculation in mice. In vitro, Mf4/4 spleen macrophages supported mild virus production (10(4)-fold less than neuroblastoma), with formation of typical virions. Bone marrow-derived macrophages (BMM) were most efficient to capture virus, but new virus production was not detected. Virus-induced cell death was significantly stronger in BMM, which might have eliminated BMM with productive infection. Still, viral RNA remained detectable in the remaining BMM for at least 4 weeks. Injection of in vitro-infected Mf4/4 in the nose or brain proved efficient to propagate infection in mice, even when cells were pre-incubated with neutralizing antibodies. Surprisingly, injection of ex-vivo-infected BMM in the brain also led to lethal infection in 8 out of 12 mice. Injection of infected Mf4/4 in the muscle mostly favoured a protective antibody response. Despite that macrophages are less fit to support virus production, they can still act as a source of infectious virus upon transfer in mice. This may be relevant for screening donor organs/cells, for which RT-PCR should be preferred over the traditional antigen or virus isolation assays.

A non-invasive intranasal inoculation technique using isoflurane anesthesia to infect the brain of mice with rabies virus.

Methods for intranasal inoculation of viruses are often described poorly and the effects of variations in the technique on the outcome are unknown. Standardization of protocols is key to compare studies and minimize animal use. The clinical and virological outcome of infection with rabies virus (genotypes 1 and 5) upon administration of different inoculum volumes (25, 50 and 100µl) and different anesthetic regimens were examined. Administration of 25µl of virus as a drop on both nostrils under brief superficial isoflurane anesthesia (92µl/dm(3), recovery after 85 ± 1 0s) was the most effective to infect the brain and induced 100% lethal infection 9 days later. Increasing the inoculum volume reduced infectivity significantly, with decreased viral loads in the brain and only 40% mortality. Increasing the depth of isoflurane anesthesia (230µl/dm(3)) improved the infectivity of the large-volume inoculum (90% mortality), probably because of suppression of swallow and sneeze reflexes. Compared to isoflurane anesthesia, xylazine-ketamine anesthesia reduced the infectivity of the inoculum significantly. Thus, administration of a small volume of virus on the nostrils under brief gas anesthesia is a safe and reproducible technique to induce infection of the brain. Since needles are not required, this helps to preserve the integrity of the physical barriers, animal welfare and the manipulator's safety.

Effect of PstS sub-units or PknD deficiency on the survival of Mycobacterium tuberculosis.

The membrane-associated phosphate-specific transporter (Pst) complex is composed of four different proteins: PstS, PstC, PstA and PstB. The PstS component detects and binds Pi with high affinity; the PstA and PstC form transmembrane pores for Pi entry, while PstB provides energy through ATP hydrolysis. In the Mycobacterium tuberculosis genome, four different gene clusters encode three PstS, and two of each of the other sub-units. We used RT-PCR to show that these clusters represent at least three distinct operons. The pstS3-containing operon was the only one induced by lack of environmental Pi. To study the physiologic role of the different PstS sub-units and that of another potential Pi receptor, PknD, we constructed and complemented their knockout (KO) mutants. In Sauton medium, the PstS1-3 KO grew faster than the Wt or the PknD KO. Following 24 h of complete starvation, the PstS3 or PknD deficient strains died if exposed to Pi poor conditions while the PstS1 and PstS2 KO survived and still grew faster than the Wt strain. These results suggest that PstS1-3 may play a role in the regulation of M. tuberculosis growth or metabolism while PstS3 and PknD contribute to the survival of the bacteria in phosphate poor conditions.

IS1096-mediated DNA rearrangements play a key role in genome evolution of Mycobacterium smegmatis.

The acquisition of DNA and the loss of genetic information are two important mechanisms that contribute to strain-specific differences in genome content. In this study, comparative genomics has allowed us to infer the roles of genomic rearrangement and changes in both distribution and copy number of the insertion element, IS1096, in the evolution of Mycobacterium smegmatis mc2155 from its progenitor, M. smegmatis ATCC 607. Comparative analysis revealed that the ATCC 607 genome contains only 11 IS1096 elements against the 24 reported in mc2155. As mc2155 evolved, there was a considerable expansion in the copy number of IS1096 (+13) as well as duplication of a 56-kb fragment flanked on both sides by IS1096; concurrently, a single IS1096 element and its flank were deleted. This study demonstrates that insertion sequence (IS) expansion and IS-induced rearrangements such as duplication, deletion and shuffling are major forces driving genomic diversity and evolution.

Immunogenicity and protective efficacy of a tuberculosis DNA vaccine co-expressing pro-apoptotic caspase-3.

DNA vaccination is a potent means for inducing strong cell-mediated immune responses and protective immunity against viral, bacterial and parasite pathogens in rodents. In an attempt to increase cross-presentation through apoptosis, the DNA-encoding caspase-2 prodomain followed by wild-type or catalytically inactive mutated caspase-3 was inserted into a plasmid encoding the 32 kDa mycolyl transferase (Ag85A) from Mycobacterium tuberculosis. Transient transfection showed that the mutated caspase induced slow apoptosis, normal protein expression and NF-kappaB activation while wild-type caspase induced rapid apoptosis, lower protein expression and no NF-kappaB activation. Ag85A specific antibody production was increased by co-expressing the mutated and decreased by co-expressing the wild-type caspase. Vaccination with pro-apoptotic plasmids triggered more Ag85A specific IFN-gamma producing spleen cells, and more efficient IL-2 and IFN-gamma producing memory cells in spleen and lungs after M. tuberculosis challenge. Compared to DNA-encoding secreted Ag85A, vaccination with DNA co-expressing wild-type caspase increased protection after infection with M. tuberculosis, while vaccination with plasmid co-expressing mutated caspase was not protective, possibly due to the stimulation of IL-6, IL-10 and IL-17A production.

Necrosis is associated with IL-6 production but apoptosis is not.

Due to loss of cell membrane integrity, necrotic cells passively release several cytosolic factors that can activate antigen presenting cells and other immune cells. In contrast, cells dying by apoptosis do not induce an inflammatory response. Here we show that necrotic cell death induced by several stimuli, such as TNF, anti-Fas or dsRNA, coincides with NF-kappaB-and p38MAPK-mediated upregulation and secretion of the pro-inflammatory cytokine IL-6. This event is greatly reduced or absent in conditions of apoptotic cell death induced by the same stimuli. This demonstrates that besides the capacity of necrotic cells to induce an inflammatory response due to leakage of cellular contents, necrotic dying cells themselves are involved in the expression and secretion of inflammatory cytokines. Moreover, inhibition of NF-kappaB and p38MAPK activation does not affect necrotic cell death in all conditions tested. This suggests that the activation of inflammatory pathways is distinct from the activation of necrotic cell death sensu strictu.

Protein synthesis persists during necrotic cell death.

Cell death is an intrinsic part of metazoan development and mammalian immune regulation. Whereas the molecular events orchestrating apoptosis have been characterized extensively, little is known about the biochemistry of necrotic cell death. Here, we show that, in contrast to apoptosis, the induction of necrosis does not lead to the shut down of protein synthesis. The rapid drop in protein synthesis observed in apoptosis correlates with caspase-dependent breakdown of eukaryotic translation initiation factor (eIF) 4G, activation of the double-stranded RNA-activated protein kinase PKR, and phosphorylation of its substrate eIF2-alpha. In necrosis induced by tumor necrosis factor, double-stranded RNA, or viral infection, de novo protein synthesis persists and 28S ribosomal RNA fragmentation, eIF2-alpha phosphorylation, and proteolytic activation of PKR are absent. Collectively, these results show that, in contrast to apoptotic cells, necrotic dying cells retain the opportunity to synthesize proteins.

A novel caspase-2 complex containing TRAF2 and RIP1.

The enzymatic activity of caspases is implicated in the execution of apoptosis and inflammation. Here we demonstrate a novel nonenzymatic function for caspase-2 other than its reported proteolytic role in apoptosis. Caspase-2, unlike caspase-3, -6, -7, -9, -11, -12, and -14, is a potent inducer of NF-kappaB and p38 MAPK activation in a TRAF2-mediated way. Caspase-2 interacts with TRAF1, TRAF2, and RIP1. Furthermore, we demonstrate that endogenous caspase-2 is recruited into a large and inducible protein complex, together with TRAF2 and RIP1. Structure-function analysis shows that NF-kappaB activation occurs independent of enzymatic activity of the protease and that the caspase recruitment domain of caspase-2 is sufficient for the activation of NF-kappaB and p38 MAPK. These results demonstrate the inducible assembly of a novel protein complex consisting of caspase-2, TRAF2, and RIP1 that activates NF-kappaB and p38 MAPK through the caspase recruitment domain of caspase-2 independently of its proteolytic activity.

INCA, a novel human caspase recruitment domain protein that inhibits interleukin-1beta generation.

Using in silico methods for screening the human genome for new caspase recruitment domain (CARD) proteins, we have identified INCA (Inhibitory CARD) as a protein that shares 81% identity with the prodomain of caspase-1. The INCA gene is located on chromosome 11q22 between the genes of COP/Pseudo-ICE and ICEBERG, two other CARD proteins that arose from caspase-1 gene duplications. We show that INCA mRNA is expressed in many tissues. INCA is specifically upregulated by interferon-gamma in the monocytic cell lines THP-1 and U937. INCA physically interacts with procaspase-1 and blocks the release of mature IL-1beta from LPS-stimulated macrophages. Unlike COP/Pseudo-ICE and procaspase-1, INCA does not interact with RIP2 and does not induce NF-kappaB activation. Our data show that INCA is a novel intracellular regulator of procaspase-1 activation, involved in the regulation of pro-IL-1beta processing and its release during inflammation.

Caspase-1 activates nuclear factor of the kappa-enhancer in B cells independently of its enzymatic activity.

The proteolytic activity of caspases is involved in apoptosis and inflammation. In this regard, caspase-1 is required for pro-interleukin (IL)-1beta and pro-IL-18 maturation. We report here on a novel function of caspase-1 as an activator of nuclear factor of the kappa-enhancer in B-cells (NF-kappaB) and p38 mitogen-activated protein kinase (MAPK). This function is not shared by the murine caspase-1 homologues caspase-11 and -12. In contrast to pro-IL-1beta maturation, caspase-1-induced NF-kappaB activation is not inhibited by the virus-derived caspase-1 inhibitor cytokine response modifier A and is equally induced by the enzymatically inactive caspase-1 C285A mutant. Although the general NF-kappaB-inhibiting protein A20 inhibits caspase-1-derived activation of NF-kappaB, dominant-negative forms of TRAF2 and RIP1 have no effect. We demonstrate that caspase-1 interacts with RIP2 and that dominant-negative forms of RIP2 and IkappaB kinase complex-beta inhibit caspase-1-mediated NF-kappaB activation. Structure-function analysis shows that the caspase recruitment domain of caspase-1 mediates the activation of NF-kappaB and p38 MAPK. These data demonstrate that caspase-1 contributes to inflammation by two distinct pathways: proteolysis of pro-IL-1beta, and RIP2-dependent activation of NF-kappaB and p38 MAPK mediated by the caspase recruitment domain.

Differential signaling to apoptotic and necrotic cell death by Fas-associated death domain protein FADD.

Two general pathways for cell death have been defined, apoptosis and necrosis. Previous studies in Jurkat cells have demonstrated that the Fas-associated death domain (FADD) is required for Fas-mediated signaling to apoptosis and necrosis. Here we developed L929rTA cell lines that allow Tet-on inducible expression and FK506-binding protein (FKBP)-mediated dimerization of FADD, FADD-death effector domain (FADD-DED), or FADD-death domain (FADD-DD). We show that expression and dimerization of FADD leads to necrosis. However, pretreatment of the cells with the Hsp90 inhibitor geldanamycin, which leads to proteasome-mediated degradation of receptor interacting protein 1 (RIP1), reverts FKBP-FADD-induced necrosis to apoptosis. Expression and dimerization of FADD-DD mediates necrotic cell death. We found that FADD-DD is able to bind RIP1, another protein necessary for Fas-mediated necrosis. Expression and dimerization of FADD-DED initiates apoptosis. Remarkably, in the presence of caspase inhibitors, FADD-DED mediates necrotic cell death. Coimmunoprecipitation studies revealed that FADD-DED in the absence procaspase-8 C/A is also capable of recruiting RIP1. However, when procaspase-8 C/A and RIP1 are expressed simultaneously, FADD-DED preferentially recruits procaspase-8 C/A.

Phagocytosis of necrotic cells by macrophages is phosphatidylserine dependent and does not induce inflammatory cytokine production.

Apoptotic cells are cleared by phagocytosis during development, homeostasis, and pathology. However, it is still unclear how necrotic cells are removed. We compared the phagocytic uptake by macrophages of variants of L929sA murine fibrosarcoma cells induced to die by tumor necrosis factor-induced necrosis or by Fas-mediated apoptosis. We show that apoptotic and necrotic cells are recognized and phagocytosed by macrophages, whereas living cells are not. In both cases, phagocytosis occurred through a phosphatidylserine-dependent mechanism, suggesting that externalization of phosphatidylserine is a general trigger for clearance by macrophages. However, uptake of apoptotic cells was more efficient both quantitatively and kinetically than phagocytosis of necrotic cells. Electron microscopy showed clear morphological differences in the mechanisms used by macrophages to engulf necrotic and apoptotic cells. Apoptotic cells were taken up as condensed membrane-bound particles of various sizes rather than as whole cells, whereas necrotic cells were internalized only as small cellular particles after loss of membrane integrity. Uptake of neither apoptotic nor necrotic L929 cells by macrophages modulated the expression of proinflammatory cytokines by the phagocytes.

Mechanisms of internalization of apoptotic and necrotic L929 cells by a macrophage cell line studied by electron microscopy.

Rapid and efficient phagocytic removal of dying cells is a key feature of apoptosis. In necrotic caspase-independent modes of death, the role and extent of phagocytosis is not well documented. To address this issue, we studied at the ultrastructural level the phagocytic response to dying cells in an in vitro phagocytosis assay with a mouse macrophage cell line (Mf4/4). As target cells, murine L929sAhFas cells were induced to die by TNFR1-mediated necrosis or by Fas-mediated apoptosis. Apoptotic L929sAhFas cells are taken up by complete engulfment of apoptotic bodies as single entities forming a tight-fitting phagosome, thus resembling the "zipper"-like mechanism of internalization. In contrast, primary and secondary necrotic cells were internalized by a macropinocytotic mechanism with formation of multiple ruffles by the ingesting macrophage. Ingestion of necrotic cellular material was invariably taking place after the integrity of the cell membrane was lost and did not occur as discrete particles, in contrast to apoptotic material that is surrounded by an intact membrane. Although nuclei of necrotic cells have been observed in the vicinity of macrophages, no uptake of necrotic nuclei was observed. The present report provides a basis for future studies aimed at discovering molecular pathways that precede these diverse mechanisms of uptake.

Regulation of the expression and processing of caspase-12.

Phylogenetic analysis clusters caspase-12 with the inflammatory caspases 1 and 11. We analyzed the expression of caspase-12 in mouse embryos, adult organs, and different cell types and tested the effect of interferons (IFNs) and other proinflammatory stimuli. Constitutive expression of the caspase-12 protein was restricted to certain cell types, such as epithelial cells, primary fibroblasts, and L929 fibrosarcoma cells. In fibroblasts and B16/B16 melanoma cells, caspase-12 expression is stimulated by IFN-gamma but not by IFN-alpha or -beta. The effect is increased further when IFN-gamma is combined with TNF, lipopolysaccharide (LPS), or dsRNA. These stimuli also induce caspase-1 and -11 but inhibit the expression of caspase-3 and -9. In contrast to caspase-1 and -11, no caspase-12 protein was detected in macrophages in any of these treatments. Transient overexpression of full-length caspase-12 leads to proteolytic processing of the enzyme and apoptosis. Similar processing occurs in TNF-, LPS-, Fas ligand-, and thapsigargin (Tg)-induced apoptosis. However, B16/B16 melanoma cells die when treated with the ER stress-inducing agent Tg whether they express caspase-12 or not.

Caspase-1 and caspase-8 cleave and inactivate cellular parkin.

Lesions in the parkin gene cause early onset Parkinson's disease by a loss of dopaminergic neurons, thus demonstrating a vital role for parkin in the survival of these neurons. Parkin is inactivated by caspase cleavage, and the major cleavage site is after Asp126. Caspases responsible for parkin cleavage were identified by several experimental paradigms. Transient coexpression of caspases and wild type parkin in HEK-293 cells identified caspase-1, -3, and -8 as efficient inducers of parkin cleavage whereas caspase-2, -7, -9, and -11 did not induce cleavage. A D126A parkin mutation abrogates cleavage induced by caspase-1 and -8, but not by caspase-3. In anti-Fas-treated Jurkat T cells, parkin cleavage was inhibited by caspase inhibitors hFlip and CrmA (but not by X-linked inhibitor of apoptosis (XIAP)), indicating that caspase-8 (but not caspase-3) is responsible for the parkin cleavage in this model. Moreover, induction of apoptosis in caspase-3-deficient MCF7 cells, either by caspase-1 or -8 overexpression or by tumor necrosis factor-alpha treatment, led to parkin cleavage. These results demonstrate that caspase-1 and -8 can directly cleave parkin and suggest that death receptor activation and inflammatory stress can cause loss of the ubiquitin ligase activity of parkin, thus causing accumulation of toxic parkin substrates and triggering dopaminergic cell death.

Disruption of HSP90 function reverts tumor necrosis factor-induced necrosis to apoptosis.

Triggering tumor necrosis factor receptor-1 (TNFR1) induces apoptosis in various cell lines. In contrast, stimulation of TNFR1 in L929sA leads to necrosis. Inhibition of HSP90, a chaperone for many kinases, by geldanamycin or radicicol shifted the response of L929sA cells to TNF from necrosis to apoptosis. This shift was blocked by CrmA but not by BCL-2 overexpression, suggesting that it occurred through activation of procaspase-8. Geldanamycin pretreatment led to a proteasome-dependent decrease in the levels of several TNFR1-interacting proteins including the kinases receptor-interacting protein, inhibitor of kappa B kinase-alpha, inhibitor of kappa B kinase-beta, and to a lesser extent the adaptors NF-kappaB essential modulator and tumor necrosis factor receptor-associated factor 2. As a consequence, NF-kappa B, p38MAPK, and JNK activation were abolished. No significant decrease in the levels of mitogen-activated protein kinases, adaptor proteins TNFR-associated death domain and Fas-associated death domain, or caspase-3, -8, and -9 could be detected. These results suggest that HSP90 client proteins play a crucial role in necrotic signaling. We conclude that inhibition of HSP90 may alter the composition of the TNFR1 complex, favoring the caspase-8-dependent apoptotic pathway. In the absence of geldanamycin, certain HSP90 client proteins may be preferentially recruited to the TNFR1 complex, promoting necrosis. Thus, the availability of proteins such as receptor-interacting protein, Fas-associated death domain, and caspase-8 can determine whether TNFR1 activation will lead to apoptosis or to necrosis.