A Method for Cranial Nerve XI Silencing During Surgery of the Foramen Magnum Region: Technical Case Report.

BACKGROUND AND IMPORTANCE: Skull base surgery involves the microdissection and intraoperative monitoring of cranial nerves, including cranial nerve XI (CN XI). Manipulation of CN XI can evoke brisk trapezius contraction, which in turn may disturb the surgical procedure and risk patient safety. Here we describe a method for temporarily silencing CN XI via direct intraoperative application of 1% lidocaine. CLINICAL PRESENTATION: A 41-yr-old woman presented with symptoms of elevated intracranial pressure and obstructive hydrocephalus secondary to a hemangioblastoma of the right cerebellar tonsil. A far-lateral suboccipital craniotomy was performed for resection of the lesion. During the initial stages of microdissection, vigorous trapezius contraction compromised the course of the operation. Following exposure of the cranial and cervical portions of CN XI, lidocaine was applied to the course of the exposed nerve. Within 3 min, trapezius electromyography demonstrated neuromuscular silencing, and further manipulation of CN XI did not cause shoulder movements. Approximately 30 min after lidocaine application, trapezius contractions returned, and lidocaine was again applied to re-silence CN XI. Gross total resection of the hemangioblastoma was performed during periods of CN XI inactivation, when trapezius contractions were absent. CONCLUSION: Direct application of lidocaine to CN XI temporarily silenced neuromuscular activity and prevented unwanted trapezius contraction during skull base microsurgery. This method improved operative safety and efficiency by significantly reducing patient movement due to the unavoidable manipulation of CN XI.

Role for serine protease HtrA (DegP) of Streptococcus pyogenes in the biogenesis of virulence factors SpeB and the hemolysin streptolysin S.

The serine protease HtrA is involved in the folding and maturation of secreted proteins, as well as in the degradation of proteins that misfold during secretion. Depletion of HtrA has been shown to affect the sensitivity of many organisms to thermal and environmental stresses, as well as being essential for virulence in many pathogens. In the present study, we compared the behaviors of several different HtrA mutants of the gram-positive pathogen Streptococcus pyogenes (group A streptococcus). Consistent with prior reports, insertional inactivation of htrA, the gene that encodes HtrA, resulted in a mutant that grew poorly at 37 degrees C. However, an identical phenotype was observed when a similar polar insertion was placed immediately downstream of htrA in the streptococcal chromosome, suggesting that the growth defect of the insertion mutant was not a direct result of insertional inactivation of htrA. This conclusion was supported by the observation that a nonpolar deletion mutation of htrA did not produce the growth defect. However, this mutation did affect the production of several secreted virulence factors whose biogenesis requires extensive processing. For the SpeB cysteine protease, the loss of HtrA was associated with a failure to proteolytically process the zymogen to an active protease. For the streptolysin S hemolysin, a dramatic increase in hemolytic activity resulted from the depletion of HtrA. Interestingly, HtrA-deficient mutants were not attenuated in a murine model of subcutaneous infection. These data add to the growing body of information that implies an important role for HtrA in the biogenesis of secreted proteins in gram-positive bacteria.

Role of RopB in growth phase expression of the SpeB cysteine protease of Streptococcus pyogenes.

The Rgg family of transcription regulators is widely distributed among gram-positive bacteria; however, how the members of this family control transcription is poorly understood. In the pathogen Streptococcus pyogenes, the Rgg family member RopB is required for transcription of the gene that encodes the secreted SpeB cysteine protease. Expression of the protease follows distinct kinetics that involves control of transcription in response to the growth phase. In this study, the contribution of RopB to growth phase control was examined. The gene encoding the protease (speB) and ropB are transcribed divergently from a 940-bp intergenic region. Primer extension analyses, in conjunction with reporter fusion studies, revealed that the major region controlling the transcription of both speB and ropB is adjacent to ropB and that the promoters for the two genes likely overlap. Furthermore, it was found that RopB is a DNA-binding protein that specifically binds to sequences in this control region. The interrelationship between ropB and speB expression was further reflected in the observation that transcription of ropB itself is subject to growth phase control. However, while expression of ropB from a promoter expressed during the early logarithmic phase of growth could complement a ropB deletion mutant, ectopic expression of ropB did not uncouple the expression of speB from its growth phase signal. These data implicate other factors in growth phase control and suggest that regulation of ropB expression itself is not the central mechanism of control.

Trigger factor-mediated prolyl isomerization influences maturation of the Streptococcus pyogenes cysteine protease.

Trigger factor, a ribosome-associated chaperone and peptidyl-prolyl cis-trans isomerase (PPIase), is essential for the secretion and maturation of the cysteine protease of the pathogenic gram-positive bacterium Streptococcus pyogenes. In the absence of trigger factor, the nascent protease polypeptide is not targeted to the secretory pathway. Some partial-function mutations restore targeting. However, the secreted protease does not efficiently mature into an enzymatically active form, suggesting that trigger factor has an additional role in protease biogenesis. Here, we show that, while not required for targeting, the PPIase activity of trigger factor is essential for maturation of the protease following its secretion from the bacterial cell. Site-specific mutations introduced into ropA, the gene which encodes trigger factor in S. pyogenes, produced mutant proteins deficient in PPIase activity. When these mutant alleles were used to replace the wild-type gene on the streptococcal chromosome, analysis of protease biogenesis revealed that, although the protease was secreted normally, it did not efficiently mature to an active form. Furthermore, mutation of a single proline residue in the protease prodomain suppressed the requirement for PPIase activity, suggesting that this residue is the target of trigger factor. These data support a model in which trigger factor-mediated prolyl isomerization influences the conformation of the prodomain, which in turn directs the protease into one of several alternative folding pathways.