Rodger Novak

Neuroprotection by a caspase inhibitor in acute bacterial meningitis

Half of the survivors of bacterial meningitis experience motor deficits, seizures, hearing loss or cognitive impairment, despite adequate bacterial killing by antibiotics. We demonstrate that the broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone (z-VAD-fmk) prevented hippocampal neuronal cell death and white blood cell influx into the cerebrospinal fluid compartment in experimental pneumococcal meningitis. Hippocampal neuronal death was due to apoptosis derived from the inflammatory response in the cerebrospinal fluid. Apoptosis was induced in vitro in human neurons by inflamed cerebrospinal fluid and was blocked by z-VAD-fmk. As apoptosis drives neuronal loss in pneumococcal meningitis, caspase inhibitors might provide a new therapeutic option directed specifically at reducing brain damage.

Apoptosis-Inducing Factor Mediates Microglial and Neuronal Apoptosis Caused by Pneumococcus

Streptococcus pneumoniae is the major cause of bacterial meningitis and it damages the hippocampus by inducing neuronal apoptosis. The blocking of caspases provides only partial protection in experimental meningitis, which suggests that there is an additional apoptotic pathway. A trigger of this pathway is the bacterium itself, as exposure of microglia or neurons to live pneumococci induces rapid apoptosis. In this study, apoptosis was not associated with the activation of caspases-1-10 and was not inhibited by z-VAD-fmk, a broad-spectrum caspase inhibitor. Rather, apoptosis was attributed to damage to mitochondria, which was followed by the release of apoptosis-inducing factor (AIF) from the mitochondria, large-scale DNA fragmentation, and hypodiploidy. Furthermore, intracytoplasmatic microinjection of AIF-specific antiserum markedly impaired pneumococcus-induced apoptosis. These findings indicate that AIF may play a central role in brain cell apoptosis and bacterial pathogenesis.

Isolation and Characterization of Vancomycin-Tolerant Streptococcus pneumoniae from the Cerebrospinal Fluid of a Patient Who Developed Recrudescent Meningitis

The emergence of tolerance to vancomycin has recently been reported in Streptococcus pneumoniae, the most common cause of bacterial meningitis. A vancomycin- and cephalosporin-tolerant strain of S. pneumoniae, the Tupelo strain, was isolated from the cerebrospinal fluid of a patient who then developed recrudescence of meningitis despite treatment with vancomycin and a third-generation cephalosporin. The Tupelo strain evidenced no lysis in the exponential or stationary phase of growth when exposed to vancomycin and only minimal loss of viability. Further characterization revealed normal autolysin expression, localization, and triggering by detergents, indicating that the defect leading to tolerance in the Tupelo strain is in the control pathway for triggering of autolysis. Because tolerance is a precursor phenotype to resistance and may lead to clinical failure of antibiotic therapy, these observations may have important implications for vancomycin use in infections caused by S. pneumoniae.

Penicillin tolerance genes of Streptococcus pneumoniae: the ABC-type manganese permease complex Psa

Downregulation of the major autolysin in Streptococcus pneumoniae leads to penicillin tolerance, a feature that is characterized by the ability to survive but not grow in the presence of antibiotic. Screening a library of mutants in pneumococcal surface proteins for the ability to survive 10 × minimum inhibitory concentration (MIC) of penicillin revealed over 10 candidate tolerance genes. One such mutant contained an insertion in the known gene psaA, which is part of the psa locus. This locus encodes an ABC‐type Mn permease complex. Sequence analysis of adjacent DNA extended the known genetic organization of the locus to include two new open reading frames (ORFs), psaB, which encodes an ATP‐binding protein, and psaC, which encodes a hydrophobic transmembrane protein. Mutagenesis of psaB, psaC, psaA and downstream psaD resulted in penicillin tolerance. Defective adhesion and reduced transformation efficiency, as reported previously for a psaA− mutant, were phenotypes shared by psaB −, psaC − and psaD − knockout mutants. Western blot analysis demonstrated that the set of mutants expressed RecA, but none of them showed translation of the autolysin gene, which is located downstream of recA. The addition of manganese (Mn) failed to correct the abnormal physiology. These results suggest that this ABC‐type Mn permease complex has a pleiotropic effect on pneumococcal physiology including adherence and autolysis. These are the first genes suggested as being involved in triggering autolysin. The results raise the possibility that loss of function of PsaA, by vaccine‐induced antibody for instance, may promote penicillin tolerance.

Signal transduction by a death signal peptide: uncovering the mechanism of bacterial killing by penicillin.

The binding of bactericidal antibiotics like penicillins, cephalosporins, and glycopeptides to their bacterial targets stops bacterial growth but does not directly cause cell death. A second process arising from the bacteria itself is necessary to trigger endogenous suicidal enzymes that dissolve the cell wall during autolysis. The signal and the trigger pathway for this event are completely unknown. Using S. pneumoniae as a model, we demonstrate that signal transduction via the two-component system VncR/S triggers multiple death pathways. We show that the signal sensed by VncR/S is a secreted peptide, Pep27, that initiates the cell death program. These data depict a novel model for the control of bacterial cell death.

Regulation of growth inhibition at high temperature, autolysis, transformation and adherence in Streptococcus pneumoniae by clpC.

The ClpC ATPase is a subfamily of HSP100/Clp molecular chaperones–regulators of proteolysis. By screening a library of loss of function mutants for the ability to survive treatment with penicillin, we identified the gene clpC. The corresponding protein was identified as a ClpC ATPase, sharing strong peptide sequence identity with ClpC of Bacillus subtilis, Listeria monocytogenes and Lactococcus lactis. Northern blot experiments showed that expression of clpC was induced in response to high temperature (40–42°C) versus 37°C, suggesting that ClpC is a heat shock protein. Insertional duplication mutagenesis of clpC resulted in increased tolerance to high temperature; a result in contrast to other bacterial Clp proteases. The clpC‐deficient mutant formed long chains and failed to undergo lysis after treatment with penicillin or vancomycin. The effect of the clpC mutation extended to deficiency of adherence to the human type II alveolar cells. Finally, the clpC disruption resulted in decreased genetic transformation. Western blot analysis demonstrated that the mutant failed to express pneumolysin and the choline‐binding proteins LytA, CbpA, CbpE, CbpF, CbpJ. These results suggest that the heat shock protein ClpC plays an essential complex pleiotropic role in pneumococcal physiology, including cell growth under heat stress, cell division, autolysis, adherence and transformation.

Extracellular targeting of choline‐binding proteins in Streptococcus pneumoniae by a zinc metalloprotease

A genetic‐based search for surface proteins of Streptococcus pneumoniae involved in adhesion identified a putative zinc metalloprotease (ZmpB). ZmpB shared high amino acid sequence similarities with IgA1 proteases of Gram‐positive bacteria, but ZmpB had neither IgA1 nor IgA2 protease activity. Analysis of a family of surface‐expressed proteins, the choline‐binding proteins (Cbp’s), in a zmpB‐deficient mutant demonstrated a global loss of surface expression of CbpA, CbpE, CbpF and CbpJ. CbpA was detected within the cytoplasm. The zmpB‐deficient mutant also failed to lyse with penicillin, a sign of lack of function of the Cbp LytA. Immunodetection studies revealed that the autolysin (LytA), normally located on the cell wall, was trapped in the cytoplasm colocalized with DNA and the transformation protein CinA. Trafficking of CinA and RecA to the cell membrane during genetic competence was also not observed in the zmpB‐deficient mutant. These results suggest a protease dependent regulatory mechanism governing the translocation of CinA and the Cbps LytA and CbpA of S. pneumoniae.

Intracerebral hemorrhages, fibrinolysis, and prostate carcinoma

Sirs: The causes of intracerebral hemorrhages (ICHs) are diverse. The majority of ICHs are caused by hypertension. After the exclusion of the common causes of ICH rare underlying conditions are often underdiagnosed. One is blood coagulation disturbances, which are responsible for about 5–6 % of ICHs according to 4 epidemiologic studies [1]. Blood coagulation disturbances can be induced by a broad range of diseases. An 80-year-old man had suffered from his first ICH three years previously, affecting the left parietooccipital region, resulting in a moderate hemiparesis, predominantly facio-brachial, with motor aphasia. This ICH was preceded by a minor ischemic stroke, the cause of which and that of the ICH remained unknown. A careful evaluation of possible primary disorders led to no relevant findings in his medical history. Two months before admission to our hospital, ICH occurred again at the same location. On the day of admission to our hospital the patient was still confused with regard to person, time and location. He suffered from global aphasia, ideomotor and ideatory apraxia, and central hemiparesis of the right side. Electrocardiogram and radiography of the chest were normal. Doppler ultrasonography revealed hypoplasia of the left vertebral artery; no abnormality of the intracranial vessels was found. Magnetic resonance angiography of the intracranial vessels showed no indications of angiomas, aneurysms or local stenoses. In the electroencephalogram, a previously undetected focus of slow wave abnormalities was found in the right hemisphere. Subsequent cranial MRI verified a new hemorrhagic focus in the right temporooccipital region (Fig. 1). Cerebrospinal fluid (CSF) showed a slightly elevated lymphomonocytic cell count (21/3/μl), increased total protein (1.07 g/l), and normal IgG-index (0.54). These results could be explained by a moderate disruption of the blood-brain barrier by ICH. The CSF culture was sterile. The most striking result of the first blood examination was a decrease of fibrinogen < 50 mg/dl (normal 150–350 mg/dl). Thrombocytes CE3 were also decreased (105,000/μl). Partial thromboplastin time was normal, international normalized ratio (INR) was 1.5. Clotting factor XIII was decreased to 29 % (normal 73–155 %). Further investigation of the blood coagulation system proved that there was fibrinolysis: D-dimer 12 mg/l (normal < 0.5 mg/l) and fibrin degradation products 80–100 μg/ml (normal < 10 μg/ml) were elevated due to the cleavage of fibrinogen and fibrin. Looking for the reason of the blood coagulation disturbance, an enlarged and indurated prostate, as well as a prostate specific antigen (PSA) of 22 ng/ml (normal < 4 ng/ml), highly suggestive of a prostate tumor, were found. Before performing prostate biopsy, the patient died from respiratory arrest and cardiac insufficiency. At autopsy massive thrombosis of leg and pelvic veins as well as emboli in both pulmonary main artery branches were evident. There was no tumor detectable at any other site, e. g., in the gut, lung, liver or brain. Neuropathological examination showed no signs of chronic hypertension or amyloid angiopathy. Brain vessels exhibited only slight signs of arteriosclerosis. Metastatic brain tumors were excluded. Histology verified a multilocular, little differentiated prostate LETTER TO THE EDITORS