Predictors for Functional Outcome in Patients with Aneurysmal Subarachnoid Hemorrhage Who Completed In-Hospital Rehabilitation in a Single Institution.

BACKGROUND: Although many studies evaluated independent prognosis factors of functional outcome in patients with subarachnoid hemorrhage (SAH) at a suitable time point, some patients take a long time to get functional improvement. The purpose of this study is to evaluate predictors for functional outcome in SAH patients who underwent surgical clipping and in-hospital rehabilitation in our single institution using Modified Rankin Scale (MRS) and Barthel Index (BI). METHODS: Two-hundred fifty-one SAH patients were admitted to our hospital from January 2008 to December 2017. Of them, 144 patients who diagnosed aneurysmal SAH, underwent surgical clipping within 72 hours, and completed subsequent in-hospital rehabilitation were included in this study. We explored their clinical variables and evaluated the relationships between those factors and functional outcome using MRS and BI. RESULTS: In multivariate analysis, independent prognostic factors of both MRS and BI were age, World Federation of Neurologic Surgeons grade, and symptomatic vasospasm. CONCLUSIONS: We suggest that age, SAH severity, and symptomatic vasospasm are associated with functional outcome in patients with aneurysmal SAH who completed surgical clipping and in-hospital rehabilitation.

Bacterial lipoproteins; biogenesis, sorting and quality control.

Bacterial lipoproteins are a subset of membrane proteins localized on either leaflet of the lipid bilayer. These proteins are anchored to membranes through their N-terminal lipid moiety attached to a conserved Cys. Since the protein moiety of most lipoproteins is hydrophilic, they are expected to play various roles in a hydrophilic environment outside the cytoplasmic membrane. Gram-negative bacteria such as Escherichia coli possess an outer membrane, to which most lipoproteins are sorted. The Lol pathway plays a central role in the sorting of lipoproteins to the outer membrane after lipoprotein precursors are processed to mature forms in the cytoplasmic membrane. Most lipoproteins are anchored to the inner leaflet of the outer membrane with their protein moiety in the periplasm. However, recent studies indicated that some lipoproteins further undergo topology change in the outer membrane, and play critical roles in the biogenesis and quality control of the outer membrane. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.

Novel translocation intermediate allows re-evaluation of roles of ATP, proton motive force and SecG at the late stage of preprotein translocation.

Presecretory proteins such as pOmpA are translocated across the inner membrane of Escherichia coli by Sec translocase powered by ATP and proton motive force (PMF). Translocation activity has been determined by protease protection assaying in vitro. We identified a new translocation intermediate at a late stage, which was protected by proteinase K (PK), but became PK sensitive upon urea extraction. At a late stage of pOmpA translocation driven by PMF in the presence of a nonhydrolyzable ATP analogue, the PK-protected materials arose, but were pulled back upon urea extraction, indicating that completion of translocation requires ATP hydrolysis. When inverted membrane vesicles prepared from secG-null strain (ΔSecG IMV) were used in the absence of PMF, the translocation intermediate was accumulated. When the ATP concentration was low in the absence of PMF, the translocation intermediate was also accumulated. Imposition of PMF in the presence of a low ATP concentration caused recovery of pOmpA translocation and resistance to urea extraction for SecG+ IMV, but not for ΔSecG IMV. Thus, analysis of the late translocation intermediate showed that two of three constituents, physiological concentration of ATP, PMF and SecG, are required for the catalytic cycle of preprotein translocation, that is, completion and subsequent initiation of translocation.

Lipoprotein sorting in bacteria.

Bacterial lipoproteins are synthesized as precursors in the cytoplasm and processed into mature forms on the cytoplasmic membrane. A lipid moiety attached to the N terminus anchors these proteins to the membrane surface. Many bacteria are predicted to express more than 100 lipoproteins, which play diverse functions on the cell surface. The Lol system, composed of five proteins, catalyzes the localization of Escherichia coli lipoproteins to the outer membrane. Some lipoproteins play vital roles in the sorting of other lipoproteins, lipopolysaccharides, and ß-barrel proteins to the outer membrane. On the basis of results from biochemical, genetic, and structural studies, we discuss the biogenesis of lipoproteins in bacteria, their importance in cellular functions, and the molecular mechanisms underlying efficient sorting of hydrophobic lipoproteins to the outer membrane through the hydrophilic periplasm.

Effects of disinfectants against norovirus virus-like particles predict norovirus inactivation.

Human noroviruses (NoVs) are a major cause of epidemic and sporadic acute gastroenteritis worldwide. Public and personal hygiene is one of the most important countermeasures for preventing spread of NoV infection. However, no a practicable cell culture system for NoV had been developed, initial tests of the virucidal effectiveness of anti-NoV disinfectants and sanitizers have been performed using surrogate viruses. In this study, NoV virus-like particles (VLPs) were used as a new surrogate for NoVs and a method for evaluating NoV inactivation using them developed. This method is based on morphological changes in VLPs after treatment with sodium hypochlorite. VLP specimens were found to become deformed and degraded in a concentration-dependent manner. Based on these results, the effects of sodium hypochlorite on VLPs were classified into four phases according to morphological changes and number of particles. Using the criteria thus established, the efficacy of ethanol, carbonates and alkali solutions against VLPs was evaluated. Deformation and aggregation of VLPs were observed after treatment with these disinfectants under specific conditions. To determine the degradation mechanism(s), VLPs were examined by SDS-PAGE and immunoblotting after treatment with sodium hypochlorite and ethanol. The band corresponding to the major capsid protein, VP1, was not detected after treatment with sodium hypochlorite at concentrations greater than 500 ppm, but remained after treatment with ethanol. These results suggest that VLPs have excellent potential as a surrogate marker for NoVs and can be used in initial virucidal effectiveness tests to determine the mechanism(s) of chemical agents on NoVs.

Glycolipozyme MPIase is essential for topology inversion of SecG during preprotein translocation.

Presecretory proteins are translocated across biological membranes through protein-conducting channels such as Sec61 (eukaryotes) and SecYEG (bacteria). SecA, a translocation ATPase, pushes preproteins out with dynamic structural changes through SecYEG. SecG, a subunit of the SecYEG channel possessing two transmembrane stretches (TMs), undergoes topology inversion coupled with SecA-dependent translocation. Recently, we characterized membrane protein integrase (MPIase), a glycolipozyme involved in not only protein integration into membranes but also preprotein translocation. We report here that SecG inversion occurs only when MPIase associates with SecYEG. We also found that MPIase modulates the dimer orientation of SecYEG. Cysteine-scanning mutagenesis mapped SecG TM 2 to a relatively hydrophilic environment. The dimer formation of SecG, crosslinked at TM 2, was not observed on SecG inversion, indicating that SecYEG undergoes a dynamic structural change during preprotein translocation.

Small-molecule inhibitors of gram-negative lipoprotein trafficking discovered by phenotypic screening.

In Gram-negative bacteria, lipoproteins are transported to the outer membrane by the Lol system. In this process, lipoproteins are released from the inner membrane by the ABC transporter LolCDE and passed to LolA, a diffusible periplasmic molecular chaperone. Lipoproteins are then transferred to the outer membrane receptor protein, LolB, for insertion in the outer membrane. Here we describe the discovery and characterization of novel pyridineimidazole compounds that inhibit this process. Escherichia coli mutants resistant to the pyridineimidazoles show no cross-resistance to other classes of antibiotics and map to either the LolC or LolE protein of the LolCDE transporter complex. The pyridineimidazoles were shown to inhibit the LolA-dependent release of the lipoprotein Lpp from E. coli spheroplasts. These results combined with bacterial cytological profiling are consistent with LolCDE-mediated disruption of lipoprotein targeting to the outer membrane as the mode of action of these pyridineimidazoles. The pyridineimidazoles are the first reported inhibitors of the LolCDE complex, a target which has never been exploited for therapeutic intervention. These compounds open the door to further interrogation of the outer membrane lipoprotein transport pathway as a target for antimicrobial therapy.

Novel antibacterial targets and compounds revealed by a high-throughput cell wall reporter assay.

UNLABELLED: A high-throughput phenotypic screen based on a Citrobacter freundii AmpC reporter expressed in Escherichia coli was executed to discover novel inhibitors of bacterial cell wall synthesis, an attractive, well-validated target for antibiotic intervention. Here we describe the discovery and characterization of sulfonyl piperazine and pyrazole compounds, each with novel mechanisms of action. E. coli mutants resistant to these compounds display no cross-resistance to antibiotics of other classes. Resistance to the sulfonyl piperazine maps to LpxH, which catalyzes the fourth step in the synthesis of lipid A, the outer membrane anchor of lipopolysaccharide (LPS). To our knowledge, this compound is the first reported inhibitor of LpxH. Resistance to the pyrazole compound mapped to mutations in either LolC or LolE, components of the essential LolCDE transporter complex, which is required for trafficking of lipoproteins to the outer membrane. Biochemical experiments with E. coli spheroplasts showed that the pyrazole compound is capable of inhibiting the release of lipoproteins from the inner membrane. Both of these compounds have significant promise as chemical probes to further interrogate the potential of these novel cell wall components for antimicrobial therapy. IMPORTANCE: The prevalence of antibacterial resistance, particularly among Gram-negative organisms, signals a need for novel antibacterial agents. A phenotypic screen using AmpC as a sensor for compounds that inhibit processes involved in Gram-negative envelope biogenesis led to the identification of two novel inhibitors with unique mechanisms of action targeting Escherichia coli outer membrane biogenesis. One compound inhibits the transport system for lipoprotein transport to the outer membrane, while the other compound inhibits synthesis of lipopolysaccharide. These results indicate that it is still possible to uncover new compounds with intrinsic antibacterial activity that inhibit novel targets related to the cell envelope, suggesting that the Gram-negative cell envelope still has untapped potential for therapeutic intervention.

Roles of the protruding loop of factor B essential for the localization of lipoproteins (LolB) in the anchoring of bacterial triacylated proteins to the outer membrane.

The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed.

Dissection of an ABC transporter LolCDE function analyzed by photo-crosslinking.

The envelope of Escherichia coli contains approximately 100 different species of lipoproteins, most of which are localized to the inner leaflet of the outer membrane. The localization of lipoprotein (Lol) system, consisting of five Lol proteins, is responsible for the trafficking of lipoproteins to the outer membrane. LolCDE binds to lipoproteins destined for the outer membrane and transfers them to the periplasmic chaperone LolA. Although the cryo-EM structures of E. coli LolCDE have been reported, the mechanisms by which outer membrane lipoproteins are transferred to LolA remain elusive. In this study, we investigated the interaction between LolCDE and lipoproteins using site-specific photo-crosslinking. We introduced a photo-crosslinkable amino acid into different locations across the four helices which form the central lipoprotein-binding cavity, and identified domains that crosslink with peptidoglycan-associated lipoprotein (Pal) in vivo. Using one of the derivatives containing the photo-crosslinkable amino acid, we developed an in vitro system to analyze the binding of lipoproteins to LolCDE. Our results indicate that compound 2, a LolCDE inhibitor, does not inhibit the binding of lipoproteins to LolCDE, but rather promotes the dissociation of bound lipoproteins from LolCDE.

Subarachnoid Hemorrhage From Ruptured Aneurysms at the Internal Carotid Artery-Posterior Communicating Artery Bifurcation Not Detectable on Preoperative Imaging Studies.

Since subarachnoid hemorrhage (SAH) due to the re-rupture of cerebral aneurysms severely worsens the prognosis, an accurate initial diagnosis is essential. Computed tomography (CT) and magnetic resonance imaging (MRI) usually detect aneurysmal subarachnoid hemorrhage (aSAH). However, in rare cases, its identification on CT- and MRI scans is difficult, and a cerebrospinal fluid (CSF) examination is required. We present preoperative imaging and intraoperative findings in patients whose aSAH detection necessitated a CSF examination. Of 225 aSAH patients who underwent preoperative imaging studies at our institution between April 2010 and August 2019, 3 females (1.3%, mean age 57.3 years) harbored undetectable aSAH due to the rupture of an internal carotid artery-posterior communicating artery (ICA-PcomA) aneurysm. The aneurysmal orientation was inferolateral. Intraoperatively, the anterior petroclinoid ligament hampered the detection of the aneurysms that firmly adhered to the surrounding arachnoid membrane. Sustained arterial pulsation and successive minor hemorrhage can lead to the gradual adhesion of an ICA-PcomA aneurysm to the surrounding arachnoid membrane and explain their atypical rupture undetectable on imaging studies and the development of acute subdural hematoma without SAH.

Preoperative Evaluation for Carotid Endarterectomy Using CT and MRI Fusion Images Without Contrast Media.

The usefulness of carotid endarterectomy (CEA) for carotid artery stenosis has been established even in the era of endovascular treatment. Digital subtraction angiography (DSA) and three-dimensional computed tomography angiography (3D-CTA) are used for preoperative evaluation of CEA; however, contrast agents cannot be used in patients with renal dysfunction or contrast agent allergy. Since the introduction of a three-dimensional image analysis software, SYNAPSE VINCENT (Fujifilm, Tokyo, Japan) in February 2016, we initially fused cervical CT, carotid three-dimensional time-of-flight magnetic resonance angiography, and carotid plaque imaging using 1.5 T magnetic resonance imaging to evaluate carotid artery stenosis in patients with renal dysfunction. Since then, we have gradually accumulated several cases, and at present, this fusion imaging is our first choice for preoperative evaluation of CEA instead of DSA or 3D-CTA. This evaluation method has many advantages over DSA and 3D-CTA, including the fact that it does not require contrast media. We report its usefulness, limitations, and cautions.

Multiple SecA molecules drive protein translocation across a single translocon with SecG inversion.

SecA is a translocation ATPase that drives protein translocation. D209N SecA, a dominant-negative mutant, binds ATP but is unable to hydrolyze it. This mutant was inactive to proOmpA translocation. However, it generated a translocation intermediate of 18 kDa. Further addition of wild-type SecA caused its translocation into either mature OmpA or another intermediate of 28 kDa that can be translocated into mature by a proton motive force. The addition of excess D209N SecA during translocation caused a topology inversion of SecG. Moreover, an intermediate of SecG inversion was identified when wild-type and D209N SecA were used in the same amounts. These results indicate that multiple SecA molecules drive translocation across a single translocon with SecG inversion. Here, we propose a revised model of proOmpA translocation in which a single catalytic cycle of SecA causes translocation of 10-13 kDa with ATP binding and hydrolysis, and SecG inversion is required when the next SecA cycle begins with additional ATP hydrolysis.

Defective lipoprotein sorting induces lolA expression through the Rcs stress response phosphorelay system.

The Escherichia coli LolA protein is a lipoprotein-specific chaperone that carries lipoproteins from the inner to the outer membrane. A dominant negative LolA mutant, LolA(I93C/F140C), in which both (93)Ile and (140)Phe are replaced by Cys, binds tightly to the lipoprotein-dedicated ABC transporter LolCDE complex on the inner membrane and therefore inhibits the detachment of outer membrane-specific lipoproteins from the inner membrane. We found that the expression of this mutant strongly induced lolA gene transcription. The depletion of the LolA or LolB protein also triggered lolA gene transcription, indicating that the inhibition of outer membrane lipoprotein transport triggers lolA transcription. To elucidate the mechanism, we isolated mutants that are unable to induce lolA transcription using the lacZ gene fused to the lolA promoter as a reporter and found that the Rcs phosphorelay system directly regulates lolA transcription. An outer membrane lipoprotein, RcsF, was essential for this activation, while the coactivator RcsA was dispensable. Taking the observation that an RcsF mutant localized in the inner membrane constitutively activated the Rcs phosphorelay system into consideration, the results shown here strongly suggest that correct lipoprotein sorting to the outer membrane is monitored by RcsF, which plays a key role in the Rcs stress response system.

Model of mouth-to-mouth transfer of bacterial lipoproteins through inner membrane LolC, periplasmic LolA, and outer membrane LolB.

Outer membrane-specific lipoproteins in Escherichia coli are released from the inner membrane by an ATP-binding cassette transporter, the LolCDE complex, which causes the formation of a soluble complex with a periplasmic molecular chaperone, LolA. LolA then transports lipoproteins to the outer membrane where an outer membrane receptor, LolB, incorporates lipoproteins into the outer membrane. The molecular mechanisms underlying the Lol-dependent lipoprotein sorting have been clarified in detail. However, it remained unclear how Lol factors interact with each other to conduct very efficient lipoprotein transfer in the periplasm where ATP is not available. To address this issue, a photo-reactive phenylalanine analogue, p-benzoyl-phenylalanine, was introduced at various positions of LolA and LolB, of which the overall structures are very similar and comprise an incomplete beta-barrel with a hydrophobic cavity inside. Cells expressing LolA or LolB derivatives containing the above analogue were irradiated with UV for in vivo photo-cross-linking. These analyses revealed a hot area in the same region of LolA and LolB, through which LolA and LolB interact with each other. This area is located at the entrance of the hydrophobic cavity. Moreover, this area in LolA is involved in the interaction with a membrane subunit, LolC, whereas no cross-linking occurs between LolA and the other membrane subunit, LolE, or ATP-binding subunit LolD, despite the structural similarity between LolC and LolE. The hydrophobic cavities of LolA and LolB were both found to bind lipoproteins inside. These results indicate that the transfer of lipoproteins through Lol proteins occurs in a mouth-to-mouth manner.

Overexpression of LolCDE allows deletion of the Escherichia coli gene encoding apolipoprotein N-acyltransferase.

Bacterial lipoproteins represent a subset of membrane-associated proteins that are covalently modified with lipids at the N-terminal cysteine. The final step of lipoprotein modification, N-acylation of apolipoproteins, is mediated by apolipoprotein N-acyltransferase (Lnt). Examinations with reconstituted proteoliposomes and a conditional mutant previously indicated that N-acylation of lipoproteins is required for their efficient release from the inner membrane catalyzed by LolA and LolCDE, the lipoprotein-specific chaperone and ABC transporter, respectively. Because Lnt is essential for Escherichia coli, a mutant lacking Lnt activity has not been isolated. However, we report here that lnt-null strains can be constructed when LolCDE is overproduced in strains lacking either the major outer membrane lipoprotein Lpp or transpeptidases that cross-link Lpp with peptidoglycan. Lipoproteins purified from the lnt-null strain exhibited increased mobility on SDS-PAGE compared to those from wild-type cells and could be sequenced by Edman degradation, indicating that lipoproteins in this mutant exist as apolipoproteins that lack N-acylation. Overexpression of Lpp in the lnt-null strain resulted in the accumulation of apoLpp in the inner membrane and caused growth arrest. In contrast to the release of mature Lpp in the presence of LolA and LolCDE, that of apoLpp from the inner membrane was significantly retarded. Furthermore, the amount of lipoproteins copurified with LolCDE was significantly reduced in the lnt-null strain. These results indicate that the affinity of LolCDE for apolipoprotein is very low, and therefore, overexpression of LolCDE is required for its release and sorting to the outer membrane.

A periplasmic LolA derivative with a lethal disulfide bond activates the Cpx stress response system.

LolA accommodates the acyl chains of lipoproteins in its hydrophobic cavity and shuttles between the inner and outer membranes through the hydrophilic periplasm to place lipoproteins in the outer membrane. The LolA(I93C/F140C) derivative, in which Cys replaces Ile at position 93 and Phe at position 140, strongly inhibited growth in the absence of a reducing agent because of the lethal intramolecular disulfide bond between the two Cys residues. Expression of I93C/F140C was found to activate the Cpx two-component system, which responds to cell envelope stress. The inhibition of growth by I93C/F140C was partly suppressed by overproduction of LolCDE, which is an ATP-binding cassette transporter and mediates the transfer of lipoproteins from the inner membrane to LolA. A substantial portion of the oxidized form, but not the reduced one, of I93C/F140C expressed on LolCDE overproduction was recovered in the membrane fraction, whereas wild-type LolA was localized in the periplasm even when LolCDE was overproduced. Moreover, LolCDE overproduction stabilized I93C/F140C and therefore caused an increase in its level. Taken together, these results indicate that oxidized I93C/F140C stably binds to LolCDE, which causes strong envelope stress.

Novel mutations of the LolCDE complex causing outer membrane localization of lipoproteins despite their inner membrane-retention signals.

In Gram-negative bacteria, lipoproteins are targeted to either the inner or outer membrane depending on their sorting signals. An ABC transporter LolCDE complex in Escherichia coli releases outer membrane-specific lipoproteins. Inner membrane-specific lipoproteins remain in the inner membrane because they each have a LolCDE-avoidance signal and therefore are not released by LolCDE. Only the LolC(A40P) mutation was previously found to cause outer membrane localization of lipoproteins despite their inner membrane-retention signals. Here, we isolated several new LolCDE mutants that cause outer membrane localization of lipoproteins possessing LolCDE-avoidance signals. Mutations were found in all three subunits of LolCDE, including the cytoplasmic ATPase subunit LolD. However, the extent of outer membrane sorting of inner membrane-specific lipoproteins differed depending on the mutation. Based on these observations, the molecular events underlying the recognition of lipoproteins by the LolCDE complex are discussed.

A novel complete reconstitution system for membrane integration of the simplest membrane protein.

A complete reconstitution system for membrane integration of the simplest protein was developed by means of defined factors. A mutant version of Pf3 coat protein, 3L-Pf3 coat, requires neither signal recognition particle/Sec factors nor a membrane potential for its integration into the cytoplasmic membrane of Escherichia coli. Although 3L-Pf3 coat is spontaneously integrated into liposomes composed of phospholipids, diacylglycerol completely blocks such spontaneous integrations at a physiological level. Under the conditions where spontaneous integration does not occur, 3L-Pf3 coat integration was absolutely dependent on a novel integration-stimulating factor. Combination of the PURE system, an in vitro translation system composed of the purified factors involved in translation in E. coli, with liposomes containing the highly purified integration-stimulating factor revealed multiple cycles of 3L-Pf3 coat integration, achieving the complete reconstitution of membrane integration. Based on the function of the factor, we propose that the factor is named MPIase (Membrane Protein Integrase).

A rapid method for identifying Byssochlamys and Hamigera.

Heat-resistant fungi, genera Byssochlamys, Talaromyces, Neosartorya, and Hamigera, contribute significantly to the spoilage of heat-processed acidic foods, due to the formation of heat-resistant ascospores. Here, we first evaluated the differences in the beta-tubulin gene between Byssochlamys and Hamigera and developed specific primers to identify the Byssochlamys species fulva, nivea, and spectabilis, and Hamigera. Using primers designed for B. fulva and B. nivea (B1F/1R), specific PCR products were detected for B. fulva and B. nivea, as well as B. langunculariae and B. zollerniae, two closely related species. Similarly, the Pae4F/4R-1 and H2F/2R primers produced specific PCR products for B. spectabilis and Hamigera, respectively. Using these three primer sets, strains involved in acidic food spoilage and environmental contamination were not detected. The detection limits of all primer sets were 1 ng of DNA by PCR and 10 pg of DNA by nested PCR. Each PCR assay was specific, even if the sample was contaminated 1,000-fold by other fungal DNA. Thus, this method has proved to possess an extremely high degree of specificity.