Guanylate-binding proteins convert cytosolic bacteria into caspase-4 signaling platforms.

Bacterial lipopolysaccharide triggers human caspase-4 (murine caspase-11) to cleave gasdermin-D and induce pyroptotic cell death. How lipopolysaccharide sequestered in the membranes of cytosol-invading bacteria activates caspases remains unknown. Here we show that in interferon-γ-stimulated cells guanylate-binding proteins (GBPs) assemble on the surface of Gram-negative bacteria into polyvalent signaling platforms required for activation of caspase-4. Caspase-4 activation is hierarchically controlled by GBPs; GBP1 initiates platform assembly, GBP2 and GBP4 control caspase-4 recruitment, and GBP3 governs caspase-4 activation. In response to cytosol-invading bacteria, activation of caspase-4 through the GBP platform is essential to induce gasdermin-D-dependent pyroptosis and processing of interleukin-18, thereby destroying the replicative niche for intracellular bacteria and alerting neighboring cells, respectively. Caspase-11 and GBPs epistatically protect mice against lethal bacterial challenge. Multiple antagonists of the pathway encoded by Shigella flexneri, a cytosol-adapted bacterium, provide compelling evolutionary evidence for the importance of the GBP-caspase-4 pathway in antibacterial defense.

Genomic and phenotypic analysis of SspH1 identifies a new Salmonella effector, SspH3.

Salmonella is a major foodborne pathogen and is responsible for a range of diseases. Not all Salmonella contributes to severe health outcomes as there is a large degree of genetic heterogeneity among the 2,600 serovars within the genus. This variability across Salmonella serovars is linked to numerous genetic elements that dictate virulence. While several genetic elements encode virulence factors with well-documented contributions to pathogenesis, many genetic elements implicated in Salmonella virulence remain uncharacterized. Many pathogens encode a family of E3 ubiquitin ligases that are delivered into the cells that they infect using a Type 3 Secretion System (T3SS). These effectors, known as NEL-domain E3s, were first characterized in Salmonella. Most Salmonella encodes the NEL-effectors sspH2 and slrP, whereas only a subset of Salmonella encodes sspH1. SspH1 has been shown to ubiquitinate the mammalian protein kinase PKN1, which has been reported to negatively regulate the pro-survival program Akt. We discovered that SspH1 mediates the degradation of PKN1 during infection of a macrophage cell line but that this degradation does not impact Akt signaling. Genomic analysis of a large collection of Salmonella genomes identified a putative new gene, sspH3, with homology to sspH1. SspH3 is a novel NEL-domain effector.

Fluorescence Lifetime Measurement of Prefibrillar Sickle Hemoglobin Oligomers as a Platform for Drug Discovery in Sickle Cell Disease.

The molecular origin of sickle cell disease (SCD) has been known since 1949, but treatments remain limited. We present the first high-throughput screening (HTS) platform for discovering small molecules that directly inhibit sickle hemoglobin (HbS) oligomerization and improve blood flow, potentially overcoming a long-standing bottleneck in SCD drug discovery. We show that at concentrations far below the threshold for nucleation and rapid polymerization, deoxygenated HbS forms small assemblies of multiple α2ß2 tetramers. Our HTS platform leverages high-sensitivity fluorescence lifetime measurements that monitor these temporally stable prefibrillar HbS oligomers. We show that this approach is sensitive to compounds that inhibit HbS polymerization with or without modulating hemoglobin oxygen binding affinity. We also report the results of a pilot small-molecule screen in which we discovered and validated several novel inhibitors of HbS oligomerization.

Shigella promotes major alteration of gut epithelial physiology and tissue invasion by shutting off host intracellular transport.

Intracellular trafficking pathways in eukaryotic cells are essential to maintain organelle identity and structure, and to regulate cell communication with its environment. Shigella flexneri invades and subverts the human colonic epithelium by the injection of virulence factors through a type 3 secretion system (T3SS). In this work, we report the multiple effects of two S. flexneri effectors, IpaJ and VirA, which target small GTPases of the Arf and Rab families, consequently inhibiting several intracellular trafficking pathways. IpaJ and VirA induce large-scale impairment of host protein secretion and block the recycling of surface receptors. Moreover, these two effectors decrease clathrin-dependent and -independent endocytosis. Therefore, S. flexneri infection induces a global blockage of host cell intracellular transport, affecting the exchange between cells and their external environment. The combined action of these effectors disorganizes the epithelial cell polarity, disturbs epithelial barrier integrity, promotes multiple invasion events, and enhances the pathogen capacity to penetrate into the colonic tissue in vivo.

FRET and optical trapping reveal mechanisms of actin activation of the power stroke and phosphate release in myosin V.

Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating that it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), whereas our simulations suggest that the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre- and post-power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport.

Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin.

We used transient biochemical and structural kinetics to elucidate the molecular mechanism of mavacamten, an allosteric cardiac myosin inhibitor and a prospective treatment for hypertrophic cardiomyopathy. We find that mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin not found in the single-headed S1 myosin motor fragment. We determined this by measuring cardiac myosin actin-activated and actin-independent ATPase and single-ATP turnover kinetics. A two-headed myosin fragment exhibits distinct autoinhibited ATP turnover kinetics compared with a single-headed fragment. Mavacamten enhanced this autoinhibition. It also enhanced autoinhibition of ADP release. Furthermore, actin changes the structure of the autoinhibited state by forcing myosin lever-arm rotation. Mavacamten slows this rotation in two-headed myosin but does not prevent it. We conclude that cardiac myosin is regulated in solution by an interaction between its two heads and propose that mavacamten stabilizes this state.

Converter domain mutations in myosin alter structural kinetics and motor function.

Myosins are molecular motors that use a conserved ATPase cycle to generate force. We investigated two mutations in the converter domain of myosin V (R712G and F750L) to examine how altering specific structural transitions in the motor ATPase cycle can impair myosin mechanochemistry. The corresponding mutations in the human ß-cardiac myosin gene are associated with hypertrophic and dilated cardiomyopathy, respectively. Despite similar steady-state actin-activated ATPase and unloaded in vitro motility-sliding velocities, both R712G and F750L were less able to overcome frictional loads measured in the loaded motility assay. Transient kinetic analysis and stopped-flow FRET demonstrated that the R712G mutation slowed the maximum ATP hydrolysis and recovery-stroke rate constants, whereas the F750L mutation enhanced these steps. In both mutants, the fast and slow power-stroke as well as actin-activated phosphate release rate constants were not significantly different from WT. Time-resolved FRET experiments revealed that R712G and F750L populate the pre- and post-power-stroke states with similar FRET distance and distance distribution profiles. The R712G mutant increased the mole fraction in the post-power-stroke conformation in the strong actin-binding states, whereas the F750L decreased this population in the actomyosin ADP state. We conclude that mutations in key allosteric pathways can shift the equilibrium and/or alter the activation energy associated with key structural transitions without altering the overall conformation of the pre- and post-power-stroke states. Thus, therapies designed to alter the transition between structural states may be able to rescue the impaired motor function induced by disease mutations.

Heart failure drug changes the mechanoenzymology of the cardiac myosin powerstroke.

Omecamtiv mecarbil (OM), a putative heart failure therapeutic, increases cardiac contractility. We hypothesize that it does this by changing the structural kinetics of the myosin powerstroke. We tested this directly by performing transient time-resolved FRET on a ventricular cardiac myosin biosensor. Our results demonstrate that OM stabilizes myosin's prepowerstroke structural state, supporting previous measurements showing that the drug shifts the equilibrium constant for myosin-catalyzed ATP hydrolysis toward the posthydrolysis biochemical state. OM slowed the actin-induced powerstroke, despite a twofold increase in the rate constant for actin-activated phosphate release, the biochemical step in myosin's ATPase cycle associated with force generation and the conversion of chemical energy into mechanical work. We conclude that OM alters the energetics of cardiac myosin's mechanical cycle, causing the powerstroke to occur after myosin weakly binds to actin and releases phosphate. We discuss the physiological implications for these changes.

Marine Bacteria, A Source for Alginolytic Enzyme to Disrupt Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa biofilms are typically associated with the chronic lung infection of cystic fibrosis (CF) patients and represent a major challenge for treatment. This opportunistic bacterial pathogen secretes alginate, a polysaccharide that is one of the main components of its biofilm. Targeting this major biofilm component has emerged as a tempting therapeutic strategy for tackling biofilm-associated bacterial infections. The enormous potential in genetic diversity of the marine microbial community make it a valuable resource for mining activities responsible for a broad range of metabolic processes, including the alginolytic activity responsible for degrading alginate. A collection of 36 bacterial isolates were purified from marine water based on their alginolytic activity. These isolates were identified based on their 16S rRNA gene sequences. Pseudoalteromonas sp. 1400 showed the highest alginolytic activity and was further confirmed to produce the enzyme alginate lyase. The purified alginate lyase (AlyP1400) produced by Pseudoalteromonas sp. 1400 showed a band of 23 KDa on a protein electrophoresis gel and exhibited a bifunctional lyase activity for both poly-mannuronic acid and poly-glucuronic acid degradation. A tryptic digestion of this gel band analyzed by liquid chromatography-tandem mass spectrometry confirmed high similarity to the alginate lyases in polysaccharide lyase family 18. The purified alginate lyase showed a maximum relative activity at 30 °C at a slightly acidic condition. It decreased the sodium alginate viscosity by over 90% and reduced the P. aeruginosa (strain PA14) biofilms by 69% after 24 h of incubation. The combined activity of AlyP1400 with carbenicillin or ciprofloxacin reduced the P. aeruginosa biofilm thickness, biovolume and surface area in a flow cell system. The present data revealed that AlyP1400 combined with conventional antibiotics helped to disrupt the biofilms produced by P. aeruginosa and can be used as a promising combinational therapeutic strategy.

E2~Ub conjugates regulate the kinase activity of Shigella effector OspG during pathogenesis.

Pathogenic bacteria introduce effector proteins directly into the cytosol of eukaryotic cells to promote invasion and colonization. OspG, a Shigella spp. effector kinase, plays a role in this process by helping to suppress the host inflammatory response. OspG has been reported to bind host E2 ubiquitin-conjugating enzymes activated with ubiquitin (E2~Ub), a key enzyme complex in ubiquitin transfer pathways. A co-crystal structure of the OspG/UbcH5c~Ub complex reveals that complex formation has important ramifications for the activity of both OspG and the UbcH5c~Ub conjugate. OspG is a minimal kinase domain containing only essential elements required for catalysis. UbcH5c~Ub binding stabilizes an active conformation of the kinase, greatly enhancing OspG kinase activity. In contrast, interaction with OspG stabilizes an extended, less reactive form of UbcH5c~Ub. Recognizing conserved E2 features, OspG can interact with at least ten distinct human E2s~Ub. Mouse oral infection studies indicate that E2~Ub conjugates act as novel regulators of OspG effector kinase function in eukaryotic host cells.

Direct real-time detection of the structural and biochemical events in the myosin power stroke.

A principal goal of molecular biophysics is to show how protein structural transitions explain physiology. We have developed a strategic tool, transient time-resolved FRET [(TR)(2)FRET], for this purpose and use it here to measure directly, with millisecond resolution, the structural and biochemical kinetics of muscle myosin and to determine directly how myosin's power stroke is coupled to the thermodynamic drive for force generation, actin-activated phosphate release, and the weak-to-strong actin-binding transition. We find that actin initiates the power stroke before phosphate dissociation and not after, as many models propose. This result supports a model for muscle contraction in which power output and efficiency are tuned by the distribution of myosin structural states. This technology should have wide application to other systems in which questions about the temporal coupling of allosteric structural and biochemical transitions remain unanswered.

Visualization of the type III secretion sorting platform of Shigella flexneri.

Bacterial type III secretion machines are widely used to inject virulence proteins into eukaryotic host cells. These secretion machines are evolutionarily related to bacterial flagella and consist of a large cytoplasmic complex, a transmembrane basal body, and an extracellular needle. The cytoplasmic complex forms a sorting platform essential for effector selection and needle assembly, but it remains largely uncharacterized. Here we use high-throughput cryoelectron tomography (cryo-ET) to visualize intact machines in a virulent Shigella flexneri strain genetically modified to produce minicells capable of interaction with host cells. A high-resolution in situ structure of the intact machine determined by subtomogram averaging reveals the cytoplasmic sorting platform, which consists of a central hub and six spokes, with a pod-like structure at the terminus of each spoke. Molecular modeling of wild-type and mutant machines allowed us to propose a model of the sorting platform in which the hub consists mainly of a hexamer of the Spa47 ATPase, whereas the MxiN protein comprises the spokes and the Spa33 protein forms the pods. Multiple contacts among those components are essential to align the Spa47 ATPase with the central channel of the MxiA protein export gate to form a unique nanomachine. The molecular architecture of the Shigella type III secretion machine and its sorting platform provide the structural foundation for further dissecting the mechanisms underlying type III secretion and pathogenesis and also highlight the major structural distinctions from bacterial flagella.

A Cardiomyopathy Mutation in the Myosin Essential Light Chain Alters Actomyosin Structure.

We have used site-directed time-resolved fluorescence resonance energy transfer to determine the effect of a pathological mutation in the human ventricular essential light chain (hVELC) of myosin, on the structural dynamics of the actin-myosin complex. The hVELC modulates the function of actomyosin, through the interaction of its N-terminal extension with actin and its C-terminal lobe with the myosin heavy chain. Several mutations in hVELC are associated with hypertrophic cardiomyopathy (HCM). Some biochemical effects of these mutations are known, but further insight is needed about their effects on the structural dynamics of functioning actomyosin. Therefore, we introduced the HCM mutation E56G into a single-cysteine (C16) hVELC construct and substituted it for the VELC of bovine cardiac myosin subfragment 1. Using a donor fluorescent probe on actin (at C374) and an acceptor probe on C16 of hVELC, we performed time-resolved fluorescence resonance energy transfer, directly detecting structural changes within the bound actomyosin complex during function. The E56G mutation has no significant effect on actin-activated ATPase activity or actomyosin affinity in the presence of ATP, or on the structure of the strong-binding S complex in the absence of ATP. However, in the presence of saturating ATP, where both W (prepowerstroke) and S (postpowerstroke) structural states are observed, the mutant increases the mole fraction of the S complex (increasing the duty ratio), while shifting the structure of the remaining W complex toward that of S, indicating a structural redistribution toward the strongly bound (force-generating) complex. We propose that this effect is responsible for the hypercontractile phenotype induced by this HCM mutation in myosin.

The bacterial pathogen-ubiquitin interface: lessons learned from Shigella.

Shigella species are the aetiological agents of shigellosis, a severe diarrhoeal disease that is a significant cause of morbidity and mortality worldwide. Shigellosis causes massive colonic destruction, high fever and bloody diarrhoea. Shigella pathogenesis is tightly linked to the ability of the bacterium to invade and replicate intracellularly within the colonic epithelium. Shigella uses a type 3 secretion system to deliver its effector proteins into the cytosol of infected cells. Among the repertoire of Shigella effectors, many are known to target components of the actin cytoskeleton to promote bacterial entry. An emerging alternate theme for effector function is the targeting of the host ubiquitin system. Ubiquitination is a post-translational modification restricted to eukaryotes and is involved in many essential host processes. By virtue of sheer number of ubiquitin-modulating effector proteins, it is clear that Shigella has invested heavily into subversion of the ubiquitin system. Understanding these host-pathogen interactions will inform us about the strategies used by successful pathogens and may also provide avenues for novel antimicrobial strategies.

Microtubules provide force to promote membrane uncoating in vacuolar escape for a cyto-invasive bacterial pathogen.

Intracellular bacterial pathogens gain entry to mammalian cells inside a vacuole derived from the host membrane. Some of them escape the bacteria-containing vacuole (BCV) and colonize the cytosol. Bacteria replicating within BCVs coopt the microtubule network to position it within infected cells, whereas the role of microtubules for cyto-invasive pathogens remains obscure. Here, we show that the microtubule motor cytoplasmic dynein-1 and specific activating adaptors are hijacked by the enterobacterium Shigella flexneri. These host proteins were found on infection-associated macropinosomes (IAMs) formed during Shigella internalization. We identified Rab8 and Rab13 as mediators of dynein recruitment and discovered that the Shigella effector protein IpaH7.8 promotes Rab13 retention on moving BCV membrane remnants, thereby facilitating membrane uncoating of the Shigella-containing vacuole. Moreover, the efficient unpeeling of BCV remnants contributes to a successful intercellular spread. Taken together, our work demonstrates how a bacterial pathogen subverts the intracellular transport machinery to secure a cytosolic niche.

Conserved structural mechanisms for autoinhibition in IpaH ubiquitin ligases.

The IpaH family of novel E3 ligase (NEL) enzymes occur in a variety of pathogenic and commensal bacteria that interact with eukaryotic hosts. We demonstrate that the leucine-rich repeat (LRR) substrate recognition domains of different IpaH enzymes autoinhibit the enzymatic activity of the adjacent catalytic novel E3 ligase domain by two distinct but conserved structural mechanisms. Autoinhibition is required for the in vivo biological activity of two IpaH enzymes in a eukaryotic model system. Autoinhibition was retro-engineered into a constitutively active IpaH enzyme from Yersinia pestis by introduction of single site substitutions, thereby demonstrating the conservation of autoregulatory infrastructure across the IpaH enzyme family.

Comparative study of GBP recruitment on two cytosol-dwelling pathogens, Francisella novicida and Shigella flexneri highlights differences in GBP repertoire and in GBP1 motif requirements.

Guanylate-Binding Proteins are interferon-inducible GTPases that play a key role in cell autonomous responses against intracellular pathogens. Despite sharing high sequence similarity, subtle differences among GBPs translate into functional divergences that are still largely not understood. A key GBP feature is the formation of supramolecular GBP complexes on the bacterial surface. Such complexes are observed when GBP1 binds lipopolysaccharide (LPS) from Shigella and Salmonella and further recruits GBP2-4. Here, we compared GBP recruitment on two cytosol-dwelling pathogens, Francisella novicida and S. flexneri. Francisella novicida was coated by GBP1 and GBP2 and to a lower extent by GBP4 in human macrophages. Contrary to S. flexneri, F. novicida was not targeted by GBP3, a feature independent of T6SS effectors. Multiple GBP1 features were required to promote targeting to F. novicida while GBP1 targeting to S. flexneri was much more permissive to GBP1 mutagenesis suggesting that GBP1 has multiple domains that cooperate to recognize F. novicida atypical LPS. Altogether our results indicate that the repertoire of GBPs recruited onto specific bacteria is dictated by GBP-specific features and by specific bacterial factors that remain to be identified.

RACK1 promotes Shigella flexneri actin-mediated invasion, motility, and cell-to-cell spreading.

Shigella flexneri is an intracellular bacterium that hijacks the host actin cytoskeleton to invade and disseminate within the colonic epithelium. Shigella's virulence factors induce actin polymerization, leading to bacterial uptake, actin tail formation, actin-mediated motility, and cell-to-cell spreading. Many host factors involved in the Shigella-prompted actin rearrangements remain elusive. Here, we studied the role of a host protein receptor for activated C kinase 1 (RACK1) in actin cytoskeleton dynamics and Shigella infection. We used time-lapse imaging to demonstrate that RACK1 facilitates Shigella-induced actin cytoskeleton remodeling at multiple levels during infection of epithelial cells. Silencing RACK1 expression impaired Shigella-induced rapid polymerizing structures, reducing host cell invasion, bacterial motility, and cell-to-cell spreading. In uninfected cells, RACK1 silencing reduced jasplakinolide-mediated filamentous actin aggregate formation and negatively affected actin turnover in fast polymerizing structures, such as membrane ruffles. Our findings provide a role of RACK1 in actin cytoskeleton dynamics and Shigella infection.

Expansion-assisted selective plane illumination microscopy for nanoscale imaging of centimeter-scale tissues.

Recent advances in tissue processing, labeling, and fluorescence microscopy are providing unprecedented views of the structure of cells and tissues at sub-diffraction resolutions and near single molecule sensitivity, driving discoveries in diverse fields of biology, including neuroscience. Biological tissue is organized over scales of nanometers to centimeters. Harnessing molecular imaging across three-dimensional samples on this scale requires new types of microscopes with larger fields of view and working distance, as well as higher imaging throughput. We present a new expansion-assisted selective plane illumination microscope (ExA-SPIM) with diffraction-limited and aberration-free performance over a large field of view (85 mm 2 ) and working distance (35 mm). Combined with new tissue clearing and expansion methods, the microscope allows nanoscale imaging of centimeter-scale samples, including entire mouse brains, with diffraction-limited resolutions and high contrast without sectioning. We illustrate ExA-SPIM by reconstructing individual neurons across the mouse brain, imaging cortico-spinal neurons in the macaque motor cortex, and tracing axons in human white matter.

Genome sequence of Shigella flexneri serotype 5a strain M90T Sm.

Bacteria of the genus Shigella are a major cause of death worldwide (L. von Seidlein et al., PLoS Med. 3:e353, 2006). We sequenced the genome of Shigella flexneri strain M90T Sm (serotype 5a) and compared it to the published genome sequence of S. flexneri strain 8401 (serotype 5b).