Heike Kotarsky

A role for the fibrinogen-binding regions of streptococcal M proteins in phagocytosis resistance

All virulent group A streptococcal isolates bind fibrinogen, a property that is closely linked to expression of type‐specific antiphagocytic surface molecules designated M proteins. Here we show that although the M proteins from two different strains, M1 and M5, both bind fibrinogen with high affinity, they interact with different regions in the ligand. Moreover, mapping experiments demonstrated that the fibrinogen‐binding regions in the M1 and M5 proteins are quite dissimilar at the amino acid sequence level and that they bind to different regions in the plasma protein. In spite of these differences, the fibrinogen‐binding regions of M1 and M5 could both be shown to contribute to streptococcal survival in human blood, providing evidence for the distinct function of a plasma protein interaction in bacterial pathogenesis.

Group A streptococcal phagocytosis resistance is independent of complement factor H and factor H-like protein 1 binding.

Factor H (FH) and factor H‐like protein 1 (FHL‐1) regulate complement activation through the alternative pathway. Several extracellular bacterial pathogens, prime targets for the complement system, bind FH and FHL‐1, thereby acquiring a potential mechanism for minimizing complement deposition on their surface. For group A streptococci (GAS), surface‐bound antiphagocytic M proteins mediate the interaction. To study the role of the FH–FHL‐1 interaction for complement deposition and opsonophagocytosis of GAS, we first constructed a set of truncated M5 protein variants and expressed them on the surface of a homologous M‐negative GAS strain. Binding experiments with the resulting strains demonstrated that the major FH–FHL‐1 binding is located in a 42‐amino‐acid region within the N‐terminal third of M5. Measurement of bacteria‐bound complement factor C3 after incubation in plasma showed that the presence of this region had little impact upon complement deposition through the alternative pathway. Moreover, streptococci expressing M5 proteins lacking the major FH and FHL‐1 binding sequence resisted phagocytosis in human blood as efficiently as bacteria expressing the wild‐type protein. Consequently, the data suggest that the binding of the regulators of the alternative pathway is of limited importance for GAS phagocytosis resistance.

Mitochondrial hepatopathies in the newborn period.

Mitochondrial disorders recognized in the neonatal period usually present as a metabolic crisis combined with one or several organ manifestations. Liver disorder in association with a respiratory chain deficiency may be overlooked since liver dysfunction is common in severely sick newborn infants. Lactacidosis, hypoglycemia, elevated serum transaminases and conjugated bilirubin are common signs of mitochondrial hepatopathy. Hepatosplenomegaly may occur in severe cases. A clinical picture with fetal growth restriction, postnatal lactacidosis, hypoglycemia, coagulopathy, and cholestasis, especially in combination with neurological symptoms or renal tubulopathy, should alert the neonatologist to direct investigations on mitochondrial disorder. A normal lactate level does not exclude respiratory chain defects. The most common liver manifestation caused by mutated mitochondrial DNA (deletion) is Pearson syndrome. Recently, mutations in several nuclear DNA genes have been identified that lead to mitochondrial hepatopathy, e.g. mitochondrial depletion syndrome caused by DGUOK, MPV17, SUCLG1, POLG1, or C10ORF2 mutations. A combination of lactacidosis, liver involvement, and Fanconi type renal tubulopathy is common when the complex III assembly factor BCS1L harbors mutations, the most severe disease with consistent genotype-phenotype correlation being the GRACILE syndrome. Mutations in nuclear translation factor genes (TRMU, EFG1, and EFTu) of the respiratory chain enzyme complexes have recently been identified. Diagnostic work-up of neonatal liver disorder should include assessment of function and structure of the complexes as well as mutation screening for known genes. So far, treatment is mainly symptomatic.

Mitochondrial disorders caused by mutations in respiratory chain assembly factors

Mitochondrial diseases involve the dysfunction of the oxidative phosphorylation (OXPHOS) system. This group of diseases presents with heterogeneous clinical symptoms affecting mainly organs with high energy demands. Defects in the multimeric complexes comprising the OXPHOS system have a dual genetic origin, mitochondrial or nuclear DNA. Although many nuclear DNA mutations involve genes coding for subunits of the respiratory complexes, the majority of mutations found to date affect factors that do not form part of the final complexes. These assembly factors or chaperones have multiple functions ranging from cofactor insertion to proper assembly/stability of the complexes. Although significant progress has been made in the last few years in the discovery of new assembly factors, the function of many remains elusive. Here, we describe assembly factors or chaperones that are required for respiratory chain complex assembly and their clinical relevance.

The GRACILE mutation introduced into Bcs1l causes postnatal complex III deficiency: A viable mouse model for mitochondrial hepatopathy.

Mitochondrial dysfunction is an important cause for neonatal liver disease. Disruption of genes encoding oxidative phosphorylation (OXPHOS) components usually causes embryonic lethality, and thus few disease models are available. We developed a mouse model for GRACILE syndrome, a neonatal mitochondrial disease with liver and kidney involvement, caused by a homozygous BCS1L mutation (232A>G). This gene encodes a chaperone required for incorporation of Rieske iron‐sulfur protein (RISP) into complex III of respiratory chain. Homozygous mutant mice after 3 weeks of age developed striking similarities to the human disease: growth failure, hepatic glycogen depletion, steatosis, fibrosis, and cirrhosis, as well as tubulopathy, complex III deficiency, lactacidosis, and short lifespan. BCS1L was decreased in whole liver cells and isolated mitochondria of mutants at all ages. RISP incorporation into complex III was diminished in symptomatic animals; however, in young animals complex III was correctly assembled. Complex III activity in liver, heart, and kidney of symptomatic mutants was decreased to 20%, 40%, and 40% of controls, respectively, as demonstrated with electron flux kinetics through complex III. In high‐resolution respirometry, CIII dysfunction resulted in decreased electron transport capacity through the respiratory chain under maximum substrate input. Complex I function, suggested to be dependent on a functional complex III, was, however, unaffected. Conclusion: We present the first viable model of complex III deficiency mimicking a human mitochondrial disorder. Incorporation of RISP into complex III in young homozygotes suggests another complex III assembly factor during early ontogenesis. The development of symptoms from about 3 weeks of age provides a convenient time window for studying the pathophysiology and treatment of mitochondrial hepatopathy and OXPHOS dysfunction in general. (HEPATOLOGY 2011:53:437‐447.)

Characterization of complex III deficiency and liver dysfunction in GRACILE syndrome caused by a BCS1L mutation.

A homozygous mutation in the complex III chaperone BCS1L causes GRACILE syndrome (intrauterine growth restriction, aminoaciduria, cholestasis, hepatic iron overload, lactacidosis). In control and patient fibroblasts we localized BCS1L in inner mitochondrial membranes. In patient liver, kidney, and heart BCS1L and Rieske protein levels, as well as the amount and activity of complex III, were decreased. Major histopathology was found in kidney and liver with cirrhosis and iron deposition, but of iron-related proteins only ferritin levels were high. In placenta from a GRACILE fetus, the ferrooxidases ceruloplasmin and hephaestin were upregulated suggesting association between iron overload and placental dysfunction.

Strain-specific restriction of the antiphagocytic property of group A streptococcal M proteins

ABSTRACT Group A streptococcal M proteins are type-specific virulence factors that inhibit phagocytosis. We used two M proteins, M5 and Emm22, to analyze the influence of genetic background on the properties of M proteins. Mutant strains, engineered to lack these M proteins, were complemented with genes encoding the homologous or heterologous M protein, and the complemented strains were analyzed for phagocytosis resistance. Neither the M5 nor the Emm22 protein conferred phagocytosis resistance in the heterologous background, but they did do so in the homologous background. This was not due to lack of surface expression in the heterologous background. Moreover, the M5 and Emm22 proteins expressed in heterologous background appeared to have normal structure, since they were not affected in their ability to bind different human plasma proteins. In particular, M5 or Emm22 had normal ability to bind human complement inhibitors, a property that has been implicated in phagocytosis resistance. Results similar to those obtained with M5 and Emm22 were obtained in experiments with the M6 and Emm4 proteins. Together, these data suggest that the surface expression of M protein alone may not be sufficient to confer phagocytosis resistance and consequently that strain-specific factors other than M and Emm proteins may contribute to the ability of group A streptococci to resist phagocytosis.

Metabolite profiles reveal energy failure and impaired beta-oxidation in liver of mice with complex III deficiency due to a BCS1L mutation.

Background & Aims Liver is a target organ in many mitochondrial disorders, especially if the complex III assembly factor BCS1L is mutated. To reveal disease mechanism due to such mutations, we have produced a transgenic mouse model with c.232A>G mutation in Bcs1l, the causative mutation for GRACILE syndrome. The homozygous mice develop mitochondrial hepatopathy with steatosis and fibrosis after weaning. Our aim was to assess cellular mechanisms for disease onset and progression using metabolomics. Methods With mass spectrometry we analyzed metabolite patterns in liver samples obtained from homozygotes and littermate controls of three ages. As oxidative stress might be a mechanism for mitochondrial hepatopathy, we also assessed H2O2 production and expression of antioxidants. Results Homozygotes had a similar metabolic profile at 14 days of age as controls, with the exception of slightly decreased AMP. At 24 days, when hepatocytes display first histopathological signs, increases in succinate, fumarate and AMP were found associated with impaired glucose turnover and beta-oxidation. At end stage disease after 30 days, these changes were pronounced with decreased carbohydrates, high levels of acylcarnitines and amino acids, and elevated biogenic amines, especially putrescine. Signs of oxidative stress were present in end-stage disease. Conclusions The findings suggest an early Krebs cycle defect with increases of its intermediates, which might play a role in disease onset. During disease progression, carbohydrate and fatty acid metabolism deteriorate leading to a starvation-like condition. The mouse model is valuable for further investigations on mechanisms in mitochondrial hepatopathy and for interventions.

The radical-binding lipocalin A1M binds to a Complex I subunit and protects mitochondrial structure and function.

AIMS During cell death, energy-consuming cell degradation and recycling programs are performed. Maintenance of energy delivery during cell death is therefore crucial, but the mechanisms to keep the mitochondrial functions intact during these processes are poorly understood. We have investigated the hypothesis that the heme- and radical-binding ubiquitous protein α1-microglobulin (A1M) is involved in protection of the mitochondria against oxidative insult during cell death. RESULTS Using blood cells, keratinocytes, and liver cells, we show that A1M binds with high affinity to apoptosis-induced cells and is localized to mitochondria. The mitochondrial Complex I subunit NDUFAB1 was identified as a major molecular target of the A1M binding. Furthermore, A1M was shown to inhibit the swelling of mitochondria, and to reverse the severely abrogated ATP-production of mitochondria when exposed to heme and reactive oxygen species (ROS). INNOVATION Import of the radical- and heme-binding protein A1M from the extracellular compartment confers protection of the mitochondrial structure and function during cellular insult. CONCLUSION A1M binds to a subunit of Complex I and has a role in assisting the mitochondria to maintain its energy delivery during cell death. A1M may also, at the same time, counteract and eliminate the ROS generated by the mitochondrial respiration to prevent oxidative damage to surrounding healthy tissue.

Complex III staining in blue native polyacrylamide gels.

For more than a decade now blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used for the study of the oxidative phosphorylation (OXPHOS) complexes. Catalytic activities of complexes I, II, IV and V can be assessed, after separation by gel electroforesis, by incubation of the BN-PAGE gel in specific staining solutions. However, until now, a reliable staining method for testing ubiquinol cytochrome c oxidoreductase (complex III) activity by BN-PAGE gel techniques was not available. In addition, spectrophotometric methods currently in use for detection of complex III deficiency in patients are not very sensitive. Here, we describe a newly developed diagnostic method for visualization of complex III activity by direct in-gel evaluation of ubiquinol cytochrome oxidoreductase activity. We validated the method by reporting the results in six patients with previously characterised complex III defects.