Coinfections identified from metagenomic analysis of cervical lymph nodes from tularemia patients.

BACKGROUND: Underlying coinfections may complicate infectious disease states but commonly go unnoticed because an a priori clinical suspicion is usually required so they can be detected via targeted diagnostic tools. Shotgun metagenomics is a broad diagnostic tool that can be useful for identifying multiple microbes simultaneously especially if coupled with lymph node aspirates, a clinical matrix known to house disparate pathogens. The objective of this study was to analyze the utility of this unconventional diagnostic approach (shotgun metagenomics) using clinical samples from human tularemia cases as a test model. Tularemia, caused by the bacterium Francisella tularensis, is an emerging infectious disease in Turkey. This disease commonly manifests as swelling of the lymph nodes nearest to the entry of infection. Because swollen cervical nodes are observed from many different types of human infections we used these clinical sample types to analyze the utility of shotgun metagenomics. METHODS: We conducted an unbiased molecular survey using shotgun metagenomics sequencing of DNA extracts from fine-needle aspirates of neck lymph nodes from eight tularemia patients who displayed protracted symptoms. The resulting metagenomics data were searched for microbial sequences (bacterial and viral). RESULTS: F. tularensis sequences were detected in all samples. In addition, we detected DNA of other known pathogens in three patients. Both Hepatitis B virus (HBV) and Human Parvovirus B-19 were detected in one individual and Human Parvovirus B-19 alone was detected in two other individuals. Subsequent PCR coupled with Sanger sequencing verified the metagenomics results. The HBV status was independently confirmed via serological diagnostics, despite evading notice during the initial assessment. CONCLUSION: Our data highlight that shotgun metagenomics of fine-needle lymph node aspirates is a promising clinical diagnostic strategy to identify coinfections. Given the feasibility of the diagnostic approach demonstrated here, further steps to promote integration of this type of diagnostic capability into mainstream clinical practice are warranted.

Unexpected Relations of Historical Anthrax Strain.

In 1998, it was claimed that an 80-year-old glass tube intentionally filled with Bacillus anthracis and embedded in a sugar lump as a WWI biological weapon still contained viable spores. Today, genome sequencing of three colonies isolated in 1998 and subjected to phylogenetic analysis surprisingly identified a well-known B. anthracis reference strain isolated in the United States in 1981, pointing to accidental laboratory contamination.IMPORTANCE Next-generation sequencing and subsequent phylogenetic analyses are useful and reliable tools for the classification of recent and historical samples. The reliability of sequences obtained and bioinformatic algorithms has increased in recent years, and research has uncovered the identity of a presumed bioweapon agent as a contaminant.

Transmission of methicillin-resistant Staphylococcus aureus infection through solid organ transplantation: confirmation via whole genome sequencing.

We describe two cases of donor-derived methicillin-resistant Staphylococcus aureus (MRSA) bacteremia that developed after transplantation of organs from a common donor who died from acute MRSA endocarditis. Both recipients developed recurrent MRSA infection despite appropriate antibiotic therapy, and required prolonged hospitalization and hospital readmission. Comparison of S. aureus whole genome sequence of DNA extracted from fixed donor tissue and recipients' isolates confirmed donor-derived transmission. Current guidelines emphasize the risk posed by donors with bacteremia from multidrug-resistant organisms. This investigation suggests that, particularly in the setting of donor endocarditis, even a standard course of prophylactic antibiotics may not be sufficient to prevent donor-derived infection.

Nepalese origin of cholera epidemic in Haiti.

Cholera appeared in Haiti in October 2010 for the first time in recorded history. The causative agent was quickly identified by the Haitian National Public Health Laboratory and the United States Centers for Disease Control and Prevention as Vibrio cholerae serogroup O1, serotype Ogawa, biotype El Tor. Since then, >500 000 government-acknowledged cholera cases and >7000 deaths have occurred, the largest cholera epidemic in the world, with the real death toll probably much higher. Questions of origin have been widely debated with some attributing the onset of the epidemic to climatic factors and others to human transmission. None of the evidence on origin supports climatic factors. Instead, recent epidemiological and molecular-genetic evidence point to the United Nations peacekeeping troops from Nepal as the source of cholera to Haiti, following their troop rotation in early October 2010. Such findings have important policy implications for shaping future international relief efforts.

Cells with a familial Alzheimer's disease mutation produce authentic beta-peptide.

Cells overexpressing the beta-amyloid precursor protein possessing a mutation found in familial Alzheimer's disease overproduce beta-amyloid peptide (A beta). Because these findings were based on immunological identification, we have chemically characterized the peptides produced. Purified A beta fragments from the conditioned media of these cells were found to have N-terminal sequence consistent with the A beta found in cerebral plaques. Mass spectrometric data demonstrated a series of A beta fragments consistent with those found in Alzheimer's disease (AD); the major species corresponding to A beta(1-40). Significantly, a longer fragment corresponding to A beta(1-42) was found. These findings suggest that this cellular system may be useful for mechanistic studies of A beta generation and possibly for the development of therapeutic agents to treat AD.

Lack of Alzheimer pathology after beta-amyloid protein injections in rat brain.

In order to establish a direct relationship between beta-amyloid protein (beta AP) and in vivo neurotoxicity, we made intraparenchymal injections and Alzet pump infusions of beta AP into the hippocampus and cortex of adult rats. We tested a number of synthetic beta AP peptides (beta AP 1-40, 1-38, and 25-35) and peptide controls (scrambled and reversed 1-40, and scrambled and reversed 25-35) over a wide range of concentrations and in a variety of vehicles. The rats were sacrificed from 2-35 days following the implant, and the brains examined by standard immunohistochemical and histological methods used to evaluate the pathologies associated with Alzheimer's disease. We report the lack of Alzheimer related pathology and no significant morphological differences between the beta AP peptide and the peptide and vehicle control injections. These observations indicate that the simple intraparenchymal injection of beta AP in the rat brain is not an appropriate model of Alzheimer-related neurotoxicity.

Exact cleavage site of Alzheimer amyloid precursor in neuronal PC-12 cells.

We have identified the secretory cleavage site in the Alzheimer amyloid precursor (APP) in a non-transfected neuronal cell line, using cyanogen bromide digests of APP purified from medium conditioned by PC-12 cells which were differentiated to a neuronal phenotype. The results obtained are most consistent with proteolysis of the Lys16-Leu17 bond in the beta amyloid peptide, followed by partial removal of Lys16 by a basic carboxypeptidase.

Cleavage of amyloid beta peptide during constitutive processing of its precursor.

The amyloid beta peptide (A beta P) is a small fragment of the much larger, broadly distributed amyloid precursor protein (APP). Abundant A beta P deposition in the brains of patients with Alzheimer's disease suggests that altered APP processing may represent a key pathogenic event. Direct protein structural analyses showed that constitutive processing in human embryonic kidney 293 cells cleaves APP in the interior of the A beta P, thus preventing A beta P deposition. A deficiency of this processing event may ultimately prove to be the etiological event in Alzheimer's disease that gives rise to senile plaque formation.

Lipophorin of the larval honeybee, Apis mellifera L.

Most insects have a major lipoprotein species in the blood (hemolymph) that serves to transport fat from the midgut to the storage depots in fat body cells and from the fat body to peripheral tissues. The generic name lipophorin is used for this lipoprotein. In larvae of the honeybee, Apis mellifera, a lipophorin has been found with properties that correlate well with those of the only other lipophorin reported for an immature insect, that of the tobacco hornworm, Manduca sexta. The honeybee lipophorin (Mr = 530,000) has a density of 1.13 g/ml, contains approximately 41% lipid and 59% protein, and contains two apoproteins, apoLp-I, Mr = 250,000 and apoLp-II, Mr = 80,000, both of which are glycosylated. The lipids consist predominantly of polar lipids, of which phospholipids and diacylglycerols represent 60% of the total. When the intact lipophorin is treated with trypsin, apoLp-I is rapidly proteolyzed, while apoLp-II is resistant, indicating a difference in exposure of the two apoproteins to the aqueous environment. Honeybee apoLp-II cross-reacts with antibodies to M. sexta apoLp-II, but not to anti-M. sexta apoLp-I. No cross-reactivity of honeybee apoLp-I to anti-M. sexta apoLp-I was observed.

Purification and properties of a predominantly female-specific protein from the hemolymph of the larva of the tobacco hornworm, Manduca sexta.

A major serum protein was isolated from the hemolymph of larvae of the female tobacco hornworm, Manduca sexta, just prior to metamorphosis. After 3 or 4 days, this predominantly female-specific protein is rapidly cleared from the hemolymph and taken up and stored by the fat body. This larval serum protein was purified by density gradient ultracentrifugation, gel permeation, and ion-exchange chromatography. The purified protein exhibits a single band on native gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chemical cross-linking with dimethylsuberimidate indicates a hexameric subunit arrangement for the native protein. The amino acid composition, relatively rich in methionine but poor in cysteine, was used to calculate a v = 0.75 cm3/g. Analytical ultracentrifugation experiments yielded S020,w = 16.9 S and D020,w = 3.23 X 10(-7) cm2/s. From these values Mr = 510,000, f/f0 = 1.22, and Stokes radius = 66.3 A were calculated. Immunoblotting experiments with anti-larval serum protein serum indicate a cross-reactivity with storage protein-1 of Bombyx mori. The amino acid composition and immunological data suggest that larval serum protein may be an example of a class of insect storage proteins distinct from the arylphorins, which are characterized by high content of aromatic amino acids.

The covalent protein structure of insecticyanin, a blue biliprotein from the hemolymph of the tobacco hornworm, Manduca sexta L.

The amino acid sequence has been determined for the insecticyanin from the hemolymph of the fifth instar larvae of the tobacco hornworm, Manduca sexta. The apoprotein is a single polypeptide chain of 189 amino acids, molecular weight 21,378, containing two disulfide bridges, 9-119 and 42-176. The sequence analysis was performed by automated Edman degradation of reduced and carboxymethylated insecticyanin and fragments generated therefrom by cyanogen bromide, trypsin, chymotrypsin, and Staphylococcus aureus proteinase. Most of the peptides were purified by reverse-phase high-performance liquid chromatography. A purification procedure for the isolation of insecticyanin in high yields and a simple method of determining disulfide linkages are also reported.

Insect apolipophorin III. Purification and properties.

The hemolymph of adult Manduca sexta (tobacco hornworm) contains a 17,000-dalton protein that can associate reversibly with the insect lipoprotein lipophorin. The protein is abundant in the hemolymph of the adult, but is found in larval hemolymph in only small amounts, and does not associate with larval lipophorin. On the basis of its association with adult lipophorin, we have designated the protein apolipophorin III. Apolipophorin III was dissociated from adult lipophorin by guanidinium chloride treatment and isolated by gel permeation and ion exchange chromatography. The unassociated apolipophorin III was also purified from lipophorin-free hemolymph by gel permeation, ion exchange, and lectin chromatography. Both preparations have identical isoelectric points and amino acid composition as well as the following properties. Apolipophorin III is a non-glycosylated polypeptide lacking cysteine and tryptophan. The 17,000-dalton polypeptide dimerizes in solution to a protein of Mr = 34,000.

Structural studies on lipophorin, an insect lipoprotein.

An insect high density lipoprotein, lipophorin, can be rapidly isolated from larval Manduca sexta (tobacco hornworm) hemolymph by single vertical spin density gradient ultracentrifugation. The two apolipoproteins (Mr = 245,000 and 78,000; designated apoLp-I and apoLp-II, respectively) were readily dissociated and separated in 6 M guanidine HCl by gel permeation chromatography. ApoLp-I and apoLp-II showed no immunological cross-reactivity on electrophoretic blots of sodium dodecyl sulfate-polyacrylamide gels. ApoLp-I and apoLp-II from lipophorin of adult M. sexta behaved identically to their larval counterparts. Amino acid compositions of larval apoLp-I and apoLp-II were similar except with respect to tryptophan and cysteine; apoLp-I contained 32 residues/mol of tryptophan (1.5 mol%) and 22 residues/mol (1.1 mol%) of cysteine; apoLp-II contained 2 residues/mol of tryptophan (0.2 mol%) and 14 residues/mol of cysteine (2.1 mol%). In double immunodiffusion tests, antiserum against apoLp-I or whole lipophorin strongly precipitated lipophorin, while antiserum against apoLp-II caused only minor precipitation. This indicates relatively greater exposure of apoLp-I to the aqueous environment.

The covalent structure of pig kidney fructose-1,6-bisphosphatase. Sequence of a 63-residue cyanogen bromide peptide containing a phosphorylatable serine.

Native pig kidney fructose-1,6-bisphosphatase, in contrast to the rat liver enzyme, is not a substrate of cyclic AMP-dependent protein kinase. However, the pig kidney enzyme becomes a substrate when phosphorylation is performed in 1.6 M urea, after prior unfolding in 8 M urea. A cyanogen bromide fragment containing the phosphorylation site has been isolated and the amino acid sequence of this 63-residue peptide has been determined. This peptide has the following sequence: Leu-Asp-Pro-Ala-Ile-Gly-Glu-Phe-Ile-Leu-Val-Asp-Arg-Asn-Val-Lys-le-Lys-Lys-Lys- Gly-Ser(P)-Ile-Tyr-Ser-Ile-Asn-Glu-Gly-Tyr-Ala-Lys-Glu-Phe-Asp-Pro-Ala-Ile-Thr- Glu-Tyr-Ile-Glu-Arg-Lys-Lys-Phe-Pro-Pro-Asp-Asn-Ser-Ala-Pro-Tyr-Gly-Ala-Arg-Tyr -Val-Gly-Ser-Met. The amino acid sequence around the phosphorylated serine residue resembles those of other protein substrates of cyclic AMP-dependent protein kinase, but it is completely different from the phosphorylation site found in native rat liver fructose-1,6-bisphosphatase.

Structure and action of heteronemertine polypeptide toxins. Amino acid sequence of Cerebratulus lacteus toxin B-II and revised structure of toxin B-IV.

The primary structure of Cerebratulus lacteus toxin B-II has been investigated by automated Edman degradation of the reduced, carboxymethylated protein and of tryptic peptides derived from the maleylated protein. As a result of these studies, the identity of all 55 amino acid residues in the polypeptide chain has been unambiguously determined. Hydroxyproline, an amino acid not normally found in nonfibrous proteins, occupies position 10 of toxin B-II. The sequence analysis of B-II led us to re-examine the sequence of Cerebratulus toxin B-IV. The revised structure of toxin B-IV is presented herein. Like B-II, toxin B-IV contains hydroxyproline at position 10 of its sequence. Comparison of the sequences of toxins B-II and B-IV reveals a high degree of homology between these two polypeptides, particularly within the NH2-terminal two-thirds of each chain.

Phosphorylation of rat hepatic fructose-1,6-bisphosphatase and pyruvate kinase.

Fructose-1,6-bisphosphatase from rat liver was phosphorylated with cyclic AMP-dependent protein kinase and [gamma-32P]ATP. Brief exposure of the 32P-labeled enzyme to trypsin removed all radioactivity from the enzyme core and produced a single-labeled peptide. The partial sequence of the 17-amino acid peptide was found to be Ser-Arg-Pro-Ser(P)-Leu-Pro-Leu-Pro-(Ser2, Glx2, Pro2, Leu, Arg2). The kinetics of cyclic AMP-dependent protein kinase-catalyzed phosphorylation of native fructose bisphosphatase were compared with those of rat liver type L pyruvate kinase where the sequence around the phosphoserine is known (Arg-Arg-Ala-Ser(P)-Val; Hjelmquist, G., Anderson, J., Edlund, B., and Engstrom, L. (1974) Biochem. Biophys. Res. Commun. 61, 559-563). The Km for pyruvate kinase (17 microM) was less than that for fructose bisphosphatase (58 microM); the Vmax was about 3-fold greater with pyruvate kinase as substrate. The relationship between the rates of phosphorylation of these native substrates and the amino acid sequences surrounding the phosphorylated sites is discussed.

The covalent structure of bovine liver rhodanese. Isolation and partial structural analysis of cyanogen bromide fragements and the complete sequence of the enzyme.

Cyanogen bromide fragments from reduced and carboxymethylated rhodanese have been isolated by gel filtration and ion exchange chromatography on columns of Sephadex G-50 and sulfoethyl-Sephadex C-25, respectively. Partial or complete structural analysis of these fragments has permitted the ordering in sequence of all eleven of the nonaligned tryptic peptides from citraconylated, S-carboxymethylcysteinyl-rhodanese and has thus provided the complete covalent structure of the enzyme. Rhodanese is a single polypeptide of 293 residues and the molecule weight calculated from the covalent structural analysis is about 32,900. The cysteinyl residue implicated in the catalytic function of rhodanese is at position 247. In some preparations of the enzyme the NH2-terminal dipeptide Val-His is missing and the sequence begins with the glutamine at position 3. The rhodanese thus obtained contains 291 amino acid residues and possesses full enzymic activity. X-ray crystallographic analysis of rhodanese has shown that the halves of the molecule (Domains I and II) are nearly identical in conformation. Comparative analysis of the sequences in Domains I and II containing residues with conformationally equivalent alpha C atoms has revealed some degree of homology between the halves of the rhodanese polypeptide. Nethertheless, the structural equivalence of the rhodanese domains is reflected much more by their similarity in tertiary structural than by their sequence homology, even when the sequence comparisons are optimized with reference to the crystallographic results.

Anthranilate synthetase component II from Pseudomonas putida. Covalent structure and identification of the cysteine residue involved in catalysis.

The complete amino acid sequence of carboxamidomethylated anthranilate synthetase component II (AS II) from Pseudomonas putida has been determined by analysis of cyanogen bromide fragments, tryptic peptides from the citraconylated protein, and by analysis of subdigests of these peptides. AS II is a single polypeptide chain of 197 residues having a calculated molecular weight of 21,684. Previous studies (Goto, Y., Keim, P. S., Zalkin, H., and Heinrikson, R. L. (1976) J. Biol. Chem, 251, 941-949) identified a cysteine residue required for the formation of an acyl-enzyme intermediate. The protein has 3 cysteine residues at positions 54, 79, and 140. Cysteine-79 was alkylated selectively by iodoacetamide and by the glutamine affinity analogue L-2-amino-4-oxo-5-chloropentanoic acid. Based on this evidence cysteine-79 is the active site residue involved in formation of the acyl-enzyme intermediate. Comparison of the P. putida AS II sequence with that of the NH2-terminal 60 residues of the enzyme from Escherichia coli shows 38% sequence identity.

Detection and kinetic behavior of preproinsulin in pancreatic islets.

Newly synthesized rat islet proteins have been analyzed by polyacrylamide slab gel electrophoresis and fluorography. A minor component having an apparent molecular weight of 11,100 was identified as preproinsulin by the sensitivity of its synthesis to glucose, the pattern of NH2-terminal leucine residues, and the rapidity of its appearance and disappearance during incubation of islets or islet cell tumors. A small amount of labeled peptide material which may represent the excised NH2-terminal extension of preproinsulin or its fragment was also detected. The kinetics of formation and processing of the preproinsulin fraction were complex, consisting of a rapidly turning over component having a half-life of about 1 min and a slower minor fraction that may have bypassed the normal cleavage process. The electrophoretic resolution of the preproinsulin and proinsulin fractions into two bands each is consistent with the presence of two closely related gene products in rat islets rather than intermediate stages in the processing of these peptides.