Elongasome core proteins and class A PBP1a display zonal, processive movement at the midcell of Streptococcus pneumoniae.

Ovoid-shaped bacteria, such as Streptococcus pneumoniae (pneumococcus), have two spatially separated peptidoglycan (PG) synthase nanomachines that locate zonally to the midcell of dividing cells. The septal PG synthase bPBP2x:FtsW closes the septum of dividing pneumococcal cells, whereas the elongasome located on the outer edge of the septal annulus synthesizes peripheral PG outward. We showed previously by sm-TIRFm that the septal PG synthase moves circumferentially at midcell, driven by PG synthesis and not by FtsZ treadmilling. The pneumococcal elongasome consists of the PG synthase bPBP2b:RodA, regulators MreC, MreD, and RodZ, but not MreB, and genetically associated proteins Class A aPBP1a and muramidase MpgA. Given its zonal location separate from FtsZ, it was of considerable interest to determine the dynamics of proteins in the pneumococcal elongasome. We found that bPBP2b, RodA, and MreC move circumferentially with the same velocities and durations at midcell, driven by PG synthesis. However, outside of the midcell zone, the majority of these elongasome proteins move diffusively over the entire surface of cells. Depletion of MreC resulted in loss of circumferential movement of bPBP2b, and bPBP2b and RodA require each other for localization and circumferential movement. Notably, a fraction of aPBP1a molecules also moved circumferentially at midcell with velocities similar to those of components of the core elongasome, but for shorter durations. Other aPBP1a molecules were static at midcell or diffusing over cell bodies. Last, MpgA displayed nonprocessive, subdiffusive motion that was largely confined to the midcell region and less frequently detected over the cell body.

Metal cofactor stabilization by a partner protein is a widespread strategy employed for amidase activation.

Construction and remodeling of the bacterial peptidoglycan (PG) cell wall must be carefully coordinated with cell growth and division. Central to cell wall construction are hydrolases that cleave bonds in peptidoglycan. These enzymes also represent potential new antibiotic targets. One such hydrolase, the amidase LytH in Staphylococcus aureus, acts to remove stem peptides from PG, controlling where substrates are available for insertion of new PG strands and consequently regulating cell size. When it is absent, cells grow excessively large and have division defects. For activity, LytH requires a protein partner, ActH, that consists of an intracellular domain, a large rhomboid protease domain, and three extracellular tetratricopeptide repeats (TPRs). Here, we demonstrate that the amidase-activating function of ActH is entirely contained in its extracellular TPRs. We show that ActH binding stabilizes metals in the LytH active site and that LytH metal binding in turn is needed for stable complexation with ActH. We further present a structure of a complex of the extracellular domains of LytH and ActH. Our findings suggest that metal cofactor stabilization is a general strategy used by amidase activators and that ActH houses multiple functions within a single protein.

Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall.

The peptidoglycan cell wall is a macromolecular structure that encases bacteria and is essential for their survival. Proper assembly of the cell wall requires peptidoglycan synthases as well as membrane-bound cleavage enzymes that control where new peptidoglycan is made and inserted. Previous studies have shown that two membrane-bound proteins in Streptococcus pneumoniae, here named MpgA and MpgB, are important in maintaining cell wall integrity. MpgA was predicted to be a lytic transglycosylase based on its homology to Escherichia coli MltG, while the enzymatic activity of MpgB was unclear. Using nascent peptidoglycan substrates synthesized in vitro from the peptidoglycan precursor Lipid II, we report that both MpgA and MpgB are muramidases. We show that replacing a single amino acid in E. coli MltG with the corresponding amino acid from MpgA results in muramidase activity, allowing us to predict from the presence of this amino acid that other putative lytic transglycosylases actually function as muramidases. Strikingly, we report that MpgA and MpgB cut nascent peptidoglycan at different positions along the sugar backbone relative to the reducing end, with MpgA producing much longer peptidoglycan oligomers. We show that the cleavage site selectivity of MpgA is controlled by the LysM-like subdomain, which is required for its full functionality in cells. We propose that MltG's ability to complement the loss of MpgA in S. pneumoniae despite performing different cleavage chemistry is because it can cleave nascent peptidoglycan at the same distance from the lipid anchor.

Uncovering the activities, biological roles, and regulation of bacterial cell wall hydrolases and tailoring enzymes.

Bacteria account for 1000-fold more biomass than humans. They vary widely in shape and size. The morphological diversity of bacteria is due largely to the different peptidoglycan-based cell wall structures that encase bacterial cells. Although the basic structure of peptidoglycan is highly conserved, consisting of long glycan strands that are cross-linked by short peptide chains, the mature cell wall is chemically diverse. Peptidoglycan hydrolases and cell wall-tailoring enzymes that regulate glycan strand length, the degree of cross-linking, and the addition of other modifications to peptidoglycan are central in determining the final architecture of the bacterial cell wall. Historically, it has been difficult to biochemically characterize these enzymes that act on peptidoglycan because suitable peptidoglycan substrates were inaccessible. In this review, we discuss fundamental aspects of bacterial cell wall synthesis, describe the regulation and diverse biochemical and functional activities of peptidoglycan hydrolases, and highlight recently developed methods to make and label defined peptidoglycan substrates. We also review how access to these substrates has now enabled biochemical studies that deepen our understanding of how bacterial cell wall enzymes cooperate to build a mature cell wall. Such improved understanding is critical to the development of new antibiotics that disrupt cell wall biogenesis, a process essential to the survival of bacteria.

Allosteric Inhibition of Human Ribonucleotide Reductase by dATP Entails the Stabilization of a Hexamer.

Ribonucleotide reductases (RNRs) are responsible for all de novo biosynthesis of DNA precursors in nature by catalyzing the conversion of ribonucleotides to deoxyribonucleotides. Because of its essential role in cell division, human RNR is a target for a number of anticancer drugs in clinical use. Like other class Ia RNRs, human RNR requires both a radical-generation subunit (ß) and nucleotide-binding subunit (α) for activity. Because of their complex dependence on allosteric effectors, however, the active and inactive quaternary forms of many class Ia RNRs have remained in question. Here, we present an X-ray crystal structure of the human α subunit in the presence of inhibiting levels of dATP, depicting a ring-shaped hexamer (α6) where the active sites line the inner hole. Surprisingly, our small-angle X-ray scattering (SAXS) results indicate that human α forms a similar hexamer in the presence of ATP, an activating effector. In both cases, α6 is assembled from dimers (α2) without a previously proposed tetramer intermediate (α4). However, we show with SAXS and electron microscopy that at millimolar ATP, the ATP-induced α6 can further interconvert with higher-order filaments. Differences in the dATP- and ATP-induced α6 were further examined by SAXS in the presence of the ß subunit and by activity assays as a function of ATP or dATP. Together, these results suggest that dATP-induced α6 is more stable than the ATP-induced α6 and that stabilization of this ring-shaped configuration provides a mechanism to prevent access of the ß subunit to the active site of α.

Necroptosis-inducing rhenium(V) oxo complexes.

Rhenium(V) oxo complexes of general formula [ReO(OMe)(N^N)Cl2], where N^N = 4,7-diphenyl-1,10-phenanthroline, 1, or 3,4,7,8-tetramethyl-1,10-phenanthroline, 2, effectively kill cancer cells by triggering necroptosis, a non-apoptotic form of cell death. Both complexes evoke necrosome (RIP1-RIP3)-dependent intracellular reactive oxygen species (ROS) production and propidium iodide uptake. The complexes also induce mitochondrial membrane potential depletion, a possible downstream effect of ROS production. Apparently, 1 and 2 are the first rhenium complexes to evoke cellular events consistent with programmed necrosis in cancer cells. Furthermore, 1 and 2 display low acute toxicity in C57BL/6 mice and reasonable stability in fresh human blood.

Elongasome core proteins and class A PBP1a display zonal, processive movement at the midcell of Streptococcus pneumoniae.

Ovoid-shaped bacteria, such as Streptococcus pneumoniae (pneumococcus), have two spatially separated peptidoglycan (PG) synthase nanomachines that locate zonally to the midcell of dividing cells. The septal PG synthase bPBP2x:FtsW closes the septum of dividing pneumococcal cells, whereas the elongasome located on the outer edge of the septal annulus synthesizes peripheral PG outward. We showed previously by sm-TIRFm that the septal PG synthase moves circumferentially at midcell, driven by PG synthesis and not by FtsZ treadmilling. The pneumococcal elongasome consists of the PG synthase bPBP2b:RodA, regulators MreC, MreD, and RodZ, but not MreB, and genetically associated proteins Class A aPBP1a and muramidase MpgA. Given its zonal location separate from FtsZ, it was of considerable interest to determine the dynamics of proteins in the pneumococcal elongasome. We found that bPBP2b, RodA, and MreC move circumferentially with the same velocities and durations at midcell, driven by PG synthesis. However, outside of the midcell zone, the majority of these elongasome proteins move diffusively over the entire surface of cells. Depletion of MreC resulted in loss of circumferential movement of bPBP2b, and bPBP2b and RodA require each other for localization and circumferential movement. Notably, a fraction of aPBP1a molecules also moved circumferentially at midcell with velocities similar to those of components of the core elongasome, but for shorter durations. Other aPBP1a molecules were static at midcell or diffusing over cell bodies. Last, MpgA displayed non-processive, subdiffusive motion that was largely confined to the midcell region and less frequently detected over the cell body.

Conflict and control in cortical responses to inconsistent emotional signals in a face-word Stroop.

Social communication is fraught with ambiguity. Negotiating the social world requires interpreting the affective signals we receive and often selecting between channels of conflicting affective information. The affective face-word Stroop (AFWS) provides an experimental paradigm which may identify cognitive-affective control mechanisms underpinning essential social-affective skills. Initial functional magnetic resonance imaging (fMRI) study of the AFWS identified right amygdala as driving this affective conflict and left rostral anterior cingulate cortex (rACC) as the locus of conflict control. We employed electroencephalogram (EEG) and eLORETA source localization to investigate the timing, location, and sequence of control processes when responding to affective conflict generated during the AFWS. However we designated affective word as the response target and affective face as the distractor to maximize conflict and control effects. Reaction times showed slowed responses in high vs. low control conditions, corresponding to a Rabbitt type control effect rather than the previously observed Grattan effect. Control related activation occurred in right rACC 96-118 ms post-stimulus, corresponding to the resolution of the P1 peak in the Visual Evoked Potential (VEP). Face distractors elicit right hemisphere control, while word distractors elicit left hemisphere control. Low control trials require rapid "booting up" control resources observable through VEPs. Incongruent trial activity in right fusiform face area is suppressed 118-156 ms post stimulus corresponding to onset and development of the N170 VEP component. Results are consistent with a predicted sequence of rapid early amygdala activation by affective conflict, then rACC inhibition of amygdala decreasing facilitation of affective face processing (however, amygdala activity is not observable with EEG).

Natural products that target the cell envelope.

The inexorable spread of resistance to clinically used drugs demands that we maintain a full pipeline of antibiotic candidates. As organisms have struggled to survive and compete over evolutionary history, they have developed the capacity to make a remarkably diverse array of natural products that target the cell envelope. A few have been developed for use in the clinic but most have not, and there are still an enormous number of opportunities to investigate. Substrate-binding antibiotics for Gram-positive organisms, phage-derived lysins, and outer membrane protein-targeting agents for Gram-negative organisms represent promising avenues where nature's gifts may be repurposed for use in the clinic.

Structure and reconstitution of a hydrolase complex that may release peptidoglycan from the membrane after polymerization.

Bacteria are encapsulated by a peptidoglycan cell wall that is essential for their survival1. During cell wall assembly, a lipid-linked disaccharide-peptide precursor called lipid II is polymerized and cross-linked to produce mature peptidoglycan. As lipid II is polymerized, nascent polymers remain membrane-anchored at one end, and the other end becomes cross-linked to the matrix2-4. How bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan is a long-standing question. Here, we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein that contains eight transmembrane helices form a complex that may function as a peptidoglycan release factor. The complex cleaves nascent peptidoglycan internally to produce free oligomers as well as lipid-linked oligomers that can undergo further elongation. The polytopic membrane protein, which is similar to a eukaryotic CAAX protease, controls the length of these products. A structure of the complex at a resolution of 2.6 Å shows that the membrane protein scaffolds the hydrolase to orient its active site for cleaving the glycan strand. We propose that this complex functions to detach newly synthesized peptidoglycan polymer from the cell membrane to complete integration into the cell wall matrix.

Implementation of a Proactive Pilot Health Plan-Driven Opioid Tapering Program to Decrease Chronic Opioid Use for Conditions of the Back and Spine in a Medicaid Population.

BACKGROUND: In 2016, the Oregon Health Authority and the Health Evidence Review Commission implemented guidance for Oregon Medicaid members who were taking opioids for chronic pain related to conditions of the back and spine. This guidance required that an individualized taper plan be developed and initiated by January 1, 2017, and a discontinuation date for all chronic opioid therapy of January 1, 2018. PROGRAM DESCRIPTION: This program evaluated the effect of a proactive and voluntary health plan-driven opioid tapering program on morphine equivalent daily dose (MEDD) before the implementation of governmental guidance. Two mailings were sent to the providers of the targeted members with a variety of resources to facilitate an opioid taper. Pharmacy claims were analyzed to measure member opioid use, in the form of MEDD, after the provider outreach to be compared with their MEDDs before the outreach. OBSERVATIONS: A total of 113 members met the study inclusion criteria for the second provider outreach. Of the 19 members' providers who submitted responses via fax to the health plan in response to this outreach, 6 indicated they would initiate taper plans. Of the 6 members with taper plans, 5 had decreases in MEDD (3.6%, 4.5%, 42.9%, 45.5%, and 46.1%) after the 3-month data collection period, while the sixth member had no change in MEDD. Of the 113 members, 16 members (14.2%) had a decrease in MEDD; 23 members (20.4%) had no change in MEDD; and 72 members (63.7%) had an increase in MEDD. IMPLICATIONS: This study demonstrated that when a physician agrees to enroll patients in a health-plan driven clinical program it may result in decreased opioid use as referenced by MEDD. However, the results also showed the progressive nature of opioid use in this population. While these initial taper results were promising, a larger sample size and longer follow-up duration are needed to validate long-term adherence to an opioid tapering program and confirm that these results are attributable to the program and not other factors. DISCLOSURES: This study was sponsored by Moda Health. Patel is employed by Moda Health; Page and Saliba were employed by Moda Health during this project; and Traver was employed by Moda Health during part of this project. Page is now employed by Oregon State University (during the writing of this manuscript) to support the College of Pharmacy's contract with the Oregon Health Authority to provide professional pharmacist support for the Oregon Medicaid program. All other authors have nothing to disclose. Study concept and design were contributed by Page and Traver, who also collected the data. Data interpretation was performed by Page and Patel. The manuscript was written by Page and revised by Page, Patel, and Saliba.