Genomic analysis of 1710 surveillance-based Neisseria gonorrhoeae isolates from the USA in 2019 identifies predominant strain types and chromosomal antimicrobial-resistance determinants.

This study characterized high-quality whole-genome sequences of a sentinel, surveillance-based collection of 1710 Neisseria gonorrhoeae (GC) isolates from 2019 collected in the USA as part of the Gonococcal Isolate Surveillance Project (GISP). It aims to provide a detailed report of strain diversity, phylogenetic relationships and resistance determinant profiles associated with reduced susceptibilities to antibiotics of concern. The 1710 isolates represented 164 multilocus sequence types and 21 predominant phylogenetic clades. Common genomic determinants defined most strains' phenotypic, reduced susceptibility to current and historic antibiotics (e.g. bla TEM plasmid for penicillin, tetM plasmid for tetracycline, gyrA for ciprofloxacin, 23S rRNA and/or mosaic mtr operon for azithromycin, and mosaic penA for cefixime and ceftriaxone). The most predominant phylogenetic clade accounted for 21 % of the isolates, included a majority of the isolates with low-level elevated MICs to azithromycin (2.0 µg ml-1), carried a mosaic mtr operon and variants in PorB, and showed expansion with respect to data previously reported from 2018. The second largest clade predominantly carried the GyrA S91F variant, was largely ciprofloxacin resistant (MIC ≥1.0 µg ml-1), and showed significant expansion with respect to 2018. Overall, a low proportion of isolates had medium- to high-level elevated MIC to azithromycin ((≥4.0 µg ml-1), based on C2611T or A2059G 23S rRNA variants). One isolate carried the penA 60.001 allele resulting in elevated MICs to cefixime and ceftriaxone of 1.0 µg ml-1. This high-resolution snapshot of genetic profiles of 1710 GC sequences, through a comparison with 2018 data (1479 GC sequences) within the sentinel system, highlights change in proportions and expansion of select GC strains and the associated genetic mechanisms of resistance. The knowledge gained through molecular surveillance may support rapid identification of outbreaks of concern. Continued monitoring may inform public health responses to limit the development and spread of antibiotic-resistant gonorrhoea.

Global Emergence and Dissemination of Neisseria gonorrhoeae ST-9363 Isolates with Reduced Susceptibility to Azithromycin.

Neisseria gonorrhoeae multilocus sequence type (ST) 9363 core-genogroup isolates have been associated with reduced azithromycin susceptibility (AZMrs) and show evidence of clonal expansion in the United States. Here, we analyze a global collection of ST-9363 core-genogroup genomes to shed light on the emergence and dissemination of this strain. The global population structure of ST-9363 core-genogroup falls into three lineages: Basal, European, and North American; with 32 clades within all lineages. Although, ST-9363 core-genogroup is inferred to have originated from Asia in the mid-19th century; we estimate the three modern lineages emerged from Europe in the late 1970s to early 1980s. The European lineage appears to have emerged and expanded from around 1986 to 1998, spreading into North America and Oceania in the mid-2000s with multiple introductions, along with multiple secondary reintroductions into Europe. Our results suggest two separate acquisition events of mosaic mtrR and mtrR promoter alleles: first during 2009-2011 and again during the 2012-2013 time, facilitating the clonal expansion of this core-genogroup with AZMrs in the United States. By tracking phylodynamic evolutionary trajectories of clades that share distinct demography as well as population-based genomic statistics, we demonstrate how recombination and selective pressures in the mtrCDE efflux operon granted a fitness advantage to establish ST-9363 as a successful gonococcal lineage in the United States and elsewhere. Although it is difficult to pinpoint the exact timing and emergence of this young core-genogroup, it remains critically important to continue monitoring it, as it could acquire additional resistance markers.

Exploring and Comparing the Structure of Sexual Networks Affected by Neisseria gonorrhoeae Using Sexual Partner Services Investigation and Genomic Data.

BACKGROUND: Sexual networks are difficult to construct because of incomplete sexual partner data. The proximity of people within a network may be inferred from genetically similar infections. We explored genomic data combined with partner services investigation (PSI) data to extend our understanding of sexual networks affected by Neisseria gonorrhoeae (NG). METHODS: We used 2017-2019 PSI and whole-genome sequencing (WGS) data from 8 jurisdictions participating in Centers for Disease Control and Prevention's Strengthening the US Response to Resistant Gonorrhea (SURRG) project. Clusters were identified from sexual contacts and through genetically similar NG isolates. Sexual mixing patterns were characterized by describing the clusters by the individual's gender and gender of their sex partners. RESULTS: Our study included 4627 diagnoses of NG infection (81% sequenced), 2455 people received a PSI, 393 people were negative contacts of cases, and 495 were contacts with an unknown NG status. We identified 823 distinct clusters using PSI data combined with WGS data. Of cases that were not linked to any other case using PSI data, 37% were linked when using WGS data. Overall, 40% of PSI cases were allocated to a larger cluster when PSI and WGS data were combined compared with PSI data alone. Mixed clusters containing women, men who report sex with women, and men who report sex with men were common when using the WGS data either alone or in combination with the PSI data. CONCLUSIONS: Combining PSI and WGS data improves our understanding of sexual network connectivity.

Genomic Analysis of the Predominant Strains and Antimicrobial Resistance Determinants Within 1479 Neisseria gonorrhoeae Isolates From the US Gonococcal Isolate Surveillance Project in 2018.

BACKGROUND: The prevalence of Neisseria gonorrhoeae (GC) isolates with elevated minimum inhibitory concentrations to various antibiotics continues to rise in the United States and globally. Genomic analysis provides a powerful tool for surveillance of circulating strains, antimicrobial resistance determinants, and understanding of transmission through a population. METHODS: Neisseria gonorrhoeae isolates collected from the US Gonococcal Isolate Surveillance Project in 2018 (n = 1479) were sequenced and characterized. Whole-genome sequencing was used to identify sequence types, antimicrobial resistance profiles, and phylogenetic relationships across demographic and geographic populations. RESULTS: Genetic characterization identified that (1) 80% of the GC isolates were represented in 33 multilocus sequence types, (2) isolates clustered in 23 major phylogenetic clusters with select phenotypic and demographic prevalence, and (3) common antimicrobial resistance determinants associated with low-level or high-level decreased susceptibility or resistance to relevant antibiotics. CONCLUSIONS: Characterization of this 2018 Gonococcal Isolate Surveillance Project genomic data set, which is the largest US whole-genome sequence data set to date, sets the basis for future prospective studies, and establishes a genomic baseline of GC populations for local and national monitoring.

Azithromycin susceptibility of Neisseria gonorrhoeae in the USA in 2017: a genomic analysis of surveillance data.

BACKGROUND: The number of cases of gonorrhoea in the USA and worldwide caused by Neisseria gonorrhoeae is increasing (555 608 reported US cases in 2017, and 87 million cases worldwide in 2016). Many countries report declining in vitro susceptibility of azithromycin, which is a concern because azithromycin and ceftriaxone are the recommended dual treatment in many countries. We aimed to identify strain types associated with decreased susceptibility to azithromycin. METHODS: We did a genomic analysis of N gonorrhoeae isolates obtained by the US Gonococcal Isolate Surveillance Project. Isolates were whole-genome sequenced based on decreased susceptibility to azithromycin (minimal inhibitory concentration [MIC] ≥2 µg/mL, using agar dilution antibiotic susceptibility testing) and geographical representation. Bioinformatic analyses established genomic diversity, strain population dynamics, and antimicrobial resistance profiles. FINDINGS: 410 isolates were sorted into more than 20 unique phylogenetic clades. One predominant persistent clade (consisting of 97 isolates) included the most isolates with azithromycin MICs of 2 µg/mL or higher (61 of 97 [63%] vs 59 of 311 [19%]; p<0·0001) and carried a mosaic mtr (multiple transferable resistance) locus (68 of 97 [70%] vs two of 313 [1%]; p<0·0001). Of the remaining 313 isolates, 57 (18%) had decreased susceptibility to azithromycin (MIC ≥4 µg/mL), which was attributed to 23S rRNA variants (56 of 57 [98%]) and formed phylogenetically diverse clades, showing various levels of clonal expansion. INTERPRETATION: Reduced azithromycin susceptibility was associated with expanding and persistent clades harbouring two well described resistance mechanisms, mosaic mtr locus and 23S rRNA variants. Understanding the role of recombination, particularly within the mtr locus, on the fitness and expansion of strains with decreased susceptibility has important implications for the public health response to minimise gonorrhoea transmission. FUNDING: US Centers for Disease Control and Prevention (CDC), CDC Combating Antibiotic Resistant Bacteria initiative, Oak Ridge Institute for Science Education, US Department of Energy/CDC/Emory University, National Institutes of Health, and Biomedical Laboratory Research and Development Service of the US Department of Veterans Affairs.

Genomic Characterization of Neisseria gonorrhoeae Strains from 2016 U.S. Sentinel Surveillance Displaying Reduced Susceptibility to Azithromycin.

In 2016, the proportion of Neisseria gonorrhoeae isolates with reduced susceptibility to azithromycin rose to 3.6%. A phylogenetic analysis of 334 N. gonorrhoeae isolates collected in 2016 revealed a single, geographically diverse lineage of isolates with MICs of 2 to 16 µg/ml that carried a mosaic-like mtr locus, whereas the majority of isolates with MICs of ≥16 µg/ml appeared sporadically and carried 23S rRNA mutations. Continued molecular surveillance of N. gonorrhoeae isolates will identify new resistance mechanisms.

Expanding U.S. Laboratory Capacity for Neisseria gonorrhoeae Antimicrobial Susceptibility Testing and Whole-Genome Sequencing through the CDC's Antibiotic Resistance Laboratory Network.

U.S. gonorrhea rates are rising, and antibiotic-resistant Neisseria gonorrhoeae (AR-Ng) is an urgent public health threat. Since implementation of nucleic acid amplification tests for N. gonorrhoeae identification, the capacity for culturing N. gonorrhoeae in the United States has declined, along with the ability to perform culture-based antimicrobial susceptibility testing (AST). Yet AST is critical for detecting and monitoring AR-Ng. In 2016, the CDC established the Antibiotic Resistance Laboratory Network (AR Lab Network) to shore up the national capacity for detecting several resistance threats including N. gonorrhoeae AR-Ng testing, a subactivity of the CDC's AR Lab Network, is performed in a tiered network of approximately 35 local laboratories, four regional laboratories (state public health laboratories in Maryland, Tennessee, Texas, and Washington), and the CDC's national reference laboratory. Local laboratories receive specimens from approximately 60 clinics associated with the Gonococcal Isolate Surveillance Project (GISP), enhanced GISP (eGISP), and the program Strengthening the U.S. Response to Resistant Gonorrhea (SURRG). They isolate and ship up to 20,000 isolates to regional laboratories for culture-based agar dilution AST with seven antibiotics and for whole-genome sequencing of up to 5,000 isolates. The CDC further examines concerning isolates and monitors genetic AR markers. During 2017 and 2018, the network tested 8,214 and 8,628 N. gonorrhoeae isolates, respectively, and the CDC received 531 and 646 concerning isolates and 605 and 3,159 sequences, respectively. In summary, the AR Lab Network supported the laboratory capacity for N. gonorrhoeae AST and associated genetic marker detection, expanding preexisting notification and analysis systems for resistance detection. Continued, robust AST and genomic capacity can help inform national public health monitoring and intervention.

Genetic Similarity of Gonococcal Homologs to Meningococcal Outer Membrane Proteins of Serogroup B Vaccine.

The human pathogens Neisseria gonorrhoeae and Neisseria meningitidis share high genome identity. Retrospective analysis of surveillance data from New Zealand indicates the potential cross-protective effect of outer membrane vesicle (OMV) meningococcal serogroup B vaccine (MeNZB) against N. gonorrhoeae A licensed OMV-based MenB vaccine, MenB-4C, consists of a recombinant FHbp, NhbA, NadA, and the MeNZB OMV. Previous work has identified several abundantly expressed outer membrane proteins (OMPs) as major components of the MenB-4C OMV with high sequence similarity between N. gonorrhoeae and N. meningitidis, suggesting a mechanism for cross-protection. To build off these findings, we performed comparative genomic analysis on 970 recent N. gonorrhoeae isolates collected through a U.S surveillance system against N. meningitidis serogroup B (NmB) reference sequences. We identified 1,525 proteins that were common to both Neisseria species, of which 57 proteins were predicted to be OMPs using in silico methods. Among the MenB-4C antigens, NhbA showed moderate sequence identity (73%) to the respective gonococcal homolog, was highly conserved within N. gonorrhoeae, and was predicted to be surface expressed. In contrast, the gonococcal FHbp was predicted not to be surface expressed, while NadA was absent in all N. gonorrhoeae isolates. Our work confirmed recent observations (E. A. Semchenko, A. Tan, R. Borrow, and K. L. Seib, Clin Infect Dis, 2018, https://doi.org/10.1093/cid/ciy1061) and describes homologous OMPs from a large panel of epidemiologically relevant N. gonorrhoeae strains in the United States against NmB reference strains. Based on our results, we report a set of OMPs that may contribute to the previously observed cross-protection and provide potential antigen targets to guide the next steps in gonorrhea vaccine development.IMPORTANCE Gonorrhea, a sexually transmitted disease, causes substantial global morbidity and economic burden. New prevention and control measures for this disease are urgently needed, as strains resistant to almost all classes of antibiotics available for treatment have emerged. Previous reports demonstrate that cross-protection from gonococcal infections may be conferred by meningococcal serogroup B (MenB) outer membrane vesicle (OMV)-based vaccines. Among 1,525 common proteins shared across the genomes of both N. gonorrhoeae and N. meningitidis, 57 proteins were predicted to be surface expressed (outer membrane proteins [OMPs]) and thus preferred targets for vaccine development. The majority of these OMPs showed high sequence identity between the 2 bacterial species. Our results provide valuable insight into the meningococcal antigens present in the current OMV-containing MenB-4C vaccine that may contribute to cross-protection against gonorrhea and may inform next steps in gonorrhea vaccine development.

Evidence of Recent Genomic Evolution in Gonococcal Strains With Decreased Susceptibility to Cephalosporins or Azithromycin in the United States, 2014-2016.

BACKGROUND: Given the lack of new antimicrobials or a vaccine, understanding the evolutionary dynamics of Neisseria gonorrhoeae is a significant public and global health priority. We investigated the emergence and spread of gonococcal strains with decreased susceptibility to cephalosporins and azithromycin using detailed genomic analyses of gonococcal isolates collected in the United States, 2014-2016. METHODS: We sequenced genomes of 649 isolates collected through the Gonococcal Isolate Surveillance Project. We examined the genetic relatedness of isolates and assessed associations between clades and various genotypic and phenotypic combinations. RESULTS: We identified a large and clonal lineage of strains (MLST ST9363) associated with elevated azithromycin minimum inhibitory concentration (AZIem), characterized by a mosaic mtr locus (C substitution in the mtrR promoter, mosaic mtrR and mtrD). Mutations in 23S rRNA were sporadically distributed among AZIem strains. Another clonal group (MLST ST1901) possessed 7 unique PBP2 patterns, and it shared common mutations in other genes associated with cephalosporin resistance. CONCLUSIONS: Whole-genome sequencing methods can enhance monitoring of antimicrobial resistant gonococcal strains by identifying gonococcal populations containing mutations of concern. These methods could inform the development of point-of-care diagnostic tests designed to determine the specific antibiotic susceptibility profile of a gonococcal infection in a patient.

Mechanistic Basis for Decreased Antimicrobial Susceptibility in a Clinical Isolate of Neisseria gonorrhoeae Possessing a Mosaic-Like mtr Efflux Pump Locus.

Recent reports suggest that mosaic-like sequences within the mtr (multiple transferable resistance) efflux pump locus of Neisseria gonorrhoeae, likely originating from commensal Neisseria sp. by transformation, can increase the ability of gonococci to resist structurally diverse antimicrobials. Thus, acquisition of numerous nucleotide changes within the mtrR gene encoding the transcriptional repressor (MtrR) of the mtrCDE efflux pump-encoding operon or overlapping promoter region for both along with those that cause amino acid changes in the MtrD transporter protein were recently reported to decrease gonococcal susceptibility to numerous antimicrobials, including azithromycin (Azi) (C. B. Wadsworth, B. J. Arnold, M. R. A. Satar, and Y. H. Grad, mBio 9:e01419-18, 2018, https://doi.org/10.1128/mBio.01419-18). We performed detailed genetic and molecular studies to define the mechanistic basis for why such strains can exhibit decreased susceptibility to MtrCDE antimicrobial substrates, including Azi. We report that a strong cis-acting transcriptional impact of a single nucleotide change within the -35 hexamer of the mtrCDE promoter as well gain-of-function amino acid changes at the C-terminal region of MtrD can mechanistically account for the decreased antimicrobial susceptibility of gonococci with a mosaic-like mtr locus.IMPORTANCE Historically, after introduction of an antibiotic for treatment of gonorrhea, strains of N. gonorrhoeae emerge that display clinical resistance due to spontaneous mutation or acquisition of resistance genes. Genetic exchange between members of the Neisseria genus occurring by transformation can cause significant changes in gonococci that impact the structure of an antibiotic target or expression of genes involved in resistance. The results presented here provide a framework for understanding how mosaic-like DNA sequences from commensal Neisseria that recombine within the gonococcal mtr efflux pump locus function to decrease bacterial susceptibility to antimicrobials, including antibiotics used in therapy of gonorrhea.

Suppressor mutations suggest a surface on PAT-4 (Integrin-linked Kinase) that interacts with UNC-112 (Kindlin).

Caenorhabditis elegans striated muscle cells attach to basement membrane and transmit the force of muscle contraction through integrin adhesion complexes. The cytoplasmic tail of ß-integrin (PAT-3) is associated with a conserved four-protein complex that includes UNC-112 (kindlin), PAT-4 (integrin-linked kinase), PAT-6 (α-parvin/actopaxin), and UNC-97 (PINCH). The proper localization of UNC-112 to muscle integrin adhesion sites requires PAT-4. A recent report (Qadota, H., Moerman, D. G., and Benian, G. M. (2012) A molecular mechanism for the requirement of PAT-4 (integrin-linked kinase (ILK)) for the localization of UNC-112 (kindlin) to integrin adhesion sites. J. Biol. Chem. 287, 28537-28551) suggests a possible molecular mechanism for this requirement: that UNC-112 exists in closed inactive and open active conformations, and conversion to the open active form is promoted by binding to PAT-4 (ILK). Previously, we also reported identification of a single missense mutation in UNC-112, D382V, which abolishes both binding to PAT-4 and normal localization to integrin adhesion sites in vivo. In this report, we describe isolation and characterization of PAT-4 missense mutations that permit binding with UNC-112 D382V and place nine affected residues on a homology model of PAT-4. These nine residues cluster in two regions on the surface of PAT-4, do not overlap the likely binding surface for PAT-6 (α-parvin), and therefore may reside along the interaction surface of PAT-4 for UNC-112 (kindlin). We also show that one of these PAT-4 mutations restores the ability of UNC-112 D382V to localize to integrin adhesions and participate in complex formation.

xMSanalyzer: automated pipeline for improved feature detection and downstream analysis of large-scale, non-targeted metabolomics data.

BACKGROUND: Detection of low abundance metabolites is important for de novo mapping of metabolic pathways related to diet, microbiome or environmental exposures. Multiple algorithms are available to extract m/z features from liquid chromatography-mass spectral data in a conservative manner, which tends to preclude detection of low abundance chemicals and chemicals found in small subsets of samples. The present study provides software to enhance such algorithms for feature detection, quality assessment, and annotation. RESULTS: xMSanalyzer is a set of utilities for automated processing of metabolomics data. The utilites can be classified into four main modules to: 1) improve feature detection for replicate analyses by systematic re-extraction with multiple parameter settings and data merger to optimize the balance between sensitivity and reliability, 2) evaluate sample quality and feature consistency, 3) detect feature overlap between datasets, and 4) characterize high-resolution m/z matches to small molecule metabolites and biological pathways using multiple chemical databases. The package was tested with plasma samples and shown to more than double the number of features extracted while improving quantitative reliability of detection. MS/MS analysis of a random subset of peaks that were exclusively detected using xMSanalyzer confirmed that the optimization scheme improves detection of real metabolites. CONCLUSIONS: xMSanalyzer is a package of utilities for data extraction, quality control assessment, detection of overlapping and unique metabolites in multiple datasets, and batch annotation of metabolites. The program was designed to integrate with existing packages such as apLCMS and XCMS, but the framework can also be used to enhance data extraction for other LC/MS data software.

Drugs that target dynamic microtubules: a new molecular perspective.

Microtubules have long been considered an ideal target for anticancer drugs because of the essential role they play in mitosis, forming the dynamic spindle apparatus. As such, there is a wide variety of compounds currently in clinical use and in development that act as antimitotic agents by altering microtubule dynamics. Although these diverse molecules are known to affect microtubule dynamics upon binding to one of the three established drug domains (taxane, vinca alkaloid, or colchicine site), the exact mechanism by which each drug works is still an area of intense speculation and research. In this study, we review the effects of microtubule-binding chemotherapeutic agents from a new perspective, considering how their mode of binding induces conformational changes and alters biological function relative to the molecular vectors of microtubule assembly or disassembly. These "biological vectors" can thus be used as a spatiotemporal context to describe molecular mechanisms by which microtubule-targeting drugs work.

Gene and protein responses of human monocytes to extracellular cysteine redox potential.

The redox potential of the major thiol/disulfide couple, cysteine (Cys) and its disulfide cystine (CySS), in plasma (E(h)Cys) is oxidized in association with oxidative stress, and oxidized E(h)Cys is associated with cardiovascular disease risk. In vitro exposure of monocytes to oxidized E(h)Cys increases expression of the proinflammatory cytokine, interleukin-1beta (IL-1beta), suggesting that E(h)Cys could be a mechanistic link between oxidative stress and chronic inflammation. Because cell membranes contain multiple Cys-rich proteins, which could be sensitive to E(h)Cys, we sought to determine whether E(h)Cys specifically affects proinflammatory signaling or has other effects on monocytes. We used microarray analysis and mass spectrometry-based proteomics to evaluate global changes in protein redox state, gene expression, and protein abundance in monocytes in response to E(h)Cys. Pathway analysis results revealed that in addition to IL-1beta-related pathways, components of stress/detoxification and cell death pathways were increased by oxidized E(h)Cys, while components of cell growth and proliferation pathways were increased by a reduced potential. Phenotypic studies confirmed that a cell stress response occurred with oxidized E(h) and that cell proliferation was stimulated with reduced E(h). Therefore, plasma E(h)Cys provides a control over monocyte phenotype, which could contribute to cardiovascular disease risk and provide a novel therapeutic target for disease prevention.

A C. elegans homolog of huntingtin-associated protein 1 is expressed in chemosensory neurons and in a number of other somatic cell types.

Huntingtin-associated protein 1 (HAP1) is a binding partner for huntingtin, the protein responsible for Huntington's disease. In mammals, HAP1 is mostly found in brain where it is expressed in neurons. Although several functions have been proposed for HAP1, its role has not yet been clearly established. In this paper, we report on the identification of a HAP1 Caenorhabditis elegans homolog called T27A3.1. T27A3.1 shows conservation with rat and human HAP1, as well as with Milton, a Drosophila HAP1 homolog. To determine the cellular expression of T27A3.1 (multiple isoforms; a-e), we generated several transgenic worm lines expressing a fluorescent reporter protein [green fluorescent protein (GFP) and DsRed2] under the control of the promoter for T27A3.1. We have found that T27A3.1 is expressed in many cell types including a subset of chemosensory neurons in the head and tail. These include the amphid chemosensory neurons ASKL and R, ASIL and R, ADFL and ASEL, the phasmid neurons PHBL and R, and the CAN neurons that are required for worm survival.

Single-molecule force spectroscopy reveals a stepwise unfolding of Caenorhabditis elegans giant protein kinase domains.

Myofibril assembly and disassembly are complex processes that regulate overall muscle mass. Titin kinase has been implicated as an initiating catalyst in signaling pathways that ultimately result in myofibril growth. In titin, the kinase domain is in an ideal position to sense mechanical strain that occurs during muscle activity. The enzyme is negatively regulated by intramolecular interactions occurring between the kinase catalytic core and autoinhibitory/regulatory region. Molecular dynamics simulations suggest that human titin kinase acts as a force sensor. However, the precise mechanism(s) resulting in the conformational changes that relieve the kinase of this autoinhibition are unknown. Here we measured the mechanical properties of the kinase domain and flanking Ig/Fn domains of the Caenorhabditis elegans titin-like proteins twitchin and TTN-1 using single-molecule atomic force microscopy. Our results show that these kinase domains have significant mechanical resistance, unfolding at forces similar to those for Ig/Fn beta-sandwich domains (30-150 pN). Further, our atomic force microscopy data is consistent with molecular dynamic simulations, which show that these kinases unfold in a stepwise fashion, first an unwinding of the autoinhibitory region, followed by a two-step unfolding of the catalytic core. These data support the hypothesis that titin kinase may function as an effective force sensor.

UNC-98 and UNC-96 interact with paramyosin to promote its incorporation into thick filaments of Caenorhabditis elegans.

Mutations in unc-96 or -98 cause reduced motility and a characteristic defect in muscle structure: by polarized light microscopy birefringent needles are found at the ends of muscle cells. Anti-paramyosin stains the needles in unc-96 and -98 mutant muscle. However there is no difference in the overall level of paramyosin in wild-type, unc-96, and -98 animals. Anti-UNC-98 and anti-paramyosin colocalize in the paramyosin accumulations of missense alleles of unc-15 (encodes paramyosin). Anti-UNC-96 and anti-UNC-98 have diffuse localization within muscles of unc-15 null mutants. By immunoblot, in the absence of paramyosin, UNC-98 is diminished, whereas in paramyosin missense mutants, UNC-98 is increased. unc-98 and -15 or unc-96 and -15 interact genetically either as double heterozygotes or as double homozygotes. By yeast two-hybrid assay and ELISAs using purified proteins, UNC-98 interacts with paramyosin residues 31-693, whereas UNC-96 interacts with a separate region of paramyosin, residues 699-798. The importance of surface charge of this 99 residue region for UNC-96 binding was shown. Paramyosin lacking the C-terminal UNC-96 binding region fails to localize throughout A-bands. We propose a model in which UNC-98 and -96 may act as chaperones to promote the incorporation of paramyosin into thick filaments.

Novel structural determinants on SIRP alpha that mediate binding to CD47.

Signal regulatory proteins (SIRP-alpha, -beta, and -gamma) are important regulators of several innate immune functions that include leukocyte migration. Membrane distal (D1) domains of SIRPalpha and SIRPgamma, but not SIRPbeta, mediate binding to a cellular ligand termed CD47. Because the extracellular domains of all SIRPs are highly homologous, we hypothesized that some of the 16 residues unique to SIRPalpha.D1 mediate binding to CD47. By site-directed mutagenesis, we determined that SIRPalpha binding to CD47 is independent of N-glycosylation. We also identified three residues critical for CD47 binding by exchanging residues on SIRPalpha with corresponding residues from SIRPbeta. Cumulative substitutions of the critical residues into SIRPbeta resulted in de novo binding of the mutant protein to CD47. Homology modeling of SIRPalpha.D1 revealed topological relationships among critical residues and allowed the identification of critical residues common to SIRPalpha and SIRPbeta. Mapping these critical residues onto the recently reported crystal structure of SIRPalpha.D1 revealed a novel region that is required for CD47 binding and is distinct and lateral to another putative CD47 binding site described on that crystal structure. The importance of this lateral region in mediating SIRPalpha.D1 binding to CD47 was confirmed by epitope mapping analyses of anti-SIRP Abs. These observations highlight a complex nature of the ligand binding requirements for SIRPalpha that appear to be dependent on two distinct but adjacent regions on the membrane distal Ig loop. A better understanding of the structural basis of SIRPalpha/CD47 interactions may provide insights into therapeutics targeting pathologic inflammation.