Monoclonal Antibodies Specific for Neisseria meningitidis Group B Polysaccharide and Their Peptide Mimotopes

ABSTRACT From five mice immunized with Escherichia coli K1 bacteria, we produced 12 immunoglobulin M hybridomas secreting monoclonal antibodies (MAbs) that bind to Neisseria meningitidis group B (NMGB). The 12 MAbs also bound the capsular polysaccharide (PS) of E. coli K1 [which, like NMGB, is α(2-8)-linked polysialic acid (PSA)] and bound to EV36, a nonpathogenic E. coli K-12 strain producing α(2-8) PSA. Except for HmenB5, which cross-reacted with N. meningitidis group C, none of the MAbs bound to N. meningitidis groups A, C, and Y. Of the 12 MAbs, 6 were autoantibodies as defined by binding to CHP-134, a neuroblastoma cell line expressing short-chain α(2-8) PSA; five of these MAbs killed NMGB in the presence of rabbit complement, and two also killed NMGB with human complement. The other six MAbs, however, were nonautoreactive; all killed NMGB with rabbit complement, and five killed NMGB with human complement. To obtain peptide mimotopes of NMGB PS, four of the nonautoreactive MAbs (HmenB2, HmenB3, HmenB13, and HmenB14) were used to screen two types of phage libraries, one with a linear peptide of 7 amino acids and the other with a circular peptide of 7 amino acids inserted between two linked cysteines. We obtained 86 phage clones that bound to the screening MAb in the absence but not in the presence of E. coli K1 PSA in solution. The clones contained 31 linear and 4 circular mimotopes expressing unique sequences. These mimotopes nonrandomly expressed amino acids and were different from previously described mimotopes for NMGB PS. The new mimotopes may be useful in producing a vaccine(s) capable of eliciting anti-NMGB antibodies not reactive with neuronal tissue.

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Tailoring a Global Iron Regulon to a Uropathogen

mBio ◽

10.1128/mbio.00351-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 1

Author(s):

Rajdeep Banerjee ◽

Erin Weisenhorn ◽

Kevin J. Schwartz ◽

Kevin S. Myers ◽

Jeremy D. Glasner ◽

...

Keyword(s):

Amino Acids ◽

Urinary Tract ◽

Amino Acid ◽

Regulatory Network ◽

Iron Homeostasis ◽

Iron Limitation ◽

Content Type ◽

E Coli ◽

General Stress ◽

K 12

ABSTRACT Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.

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#92: What Is the Optimal Management of Patients Immunosuppressed with the Anti-compliment Monoclonal Antibody, Eculizumab? A Case Report and Review

Journal of the Pediatric Infectious Diseases Society ◽

10.1093/jpids/piaa170.034 ◽

2021 ◽

Vol 10 (Supplement_1) ◽

pp. S12-S12

Author(s):

I Tinoco ◽

A Jarrell ◽

L Correa ◽

J Bissler ◽

J DeVincenzo ◽

...

Keyword(s):

Monoclonal Antibody ◽

Renal Failure ◽

Antibiotic Prophylaxis ◽

Neisseria Meningitidis ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Neisseria Species ◽

E Coli ◽

Increased Risk ◽

Prescribing Information ◽

Rabbit Complement

Abstract Background Patients with deficiencies of terminal components of complement are at hundreds to thousands fold increased risk of severe and fatal Neisseria spp. infections compared with the general population. Eculizumab is a newly approved monoclonal antibody C5 complement inhibitor. It is indicated for the treatment of atypical hemolytic uremic syndrome (atypical HUS), myasthenia gravis, and paroxysmal nocturnal hemoglobinuria. Because of the complement-depleting effect of Eculizumab dosing (Soliris®, Alexion Pharmaceuticals, Munich, Germany), patients are immunosuppressed for specific infectious pathogens (including Neisseria species) against which protection partially relies on normal complement activity. Because Eculizumab treatment is associated with a dramatically increased risk of Neisseria species. infections, recommendations for Neisseria meningitidis vaccination and antibiotic prophylaxis are contained in Eculizumab prescribing information. However, the most appropriate prevention of infections after Eculizumab has yet to be determined. Methods Case report and literature review. Results A previously healthy 7-year-old male was diagnosed with atypical HUS which included renal failure progressing to dialysis, persistent thrombocytopenia, hemolytic anemia, and hemoglobinuria. Stool cultures and a stool multiplex PCR panel did not detect Shiga-like producing E. coli nor E. coli O157/H7. Eculizumab dosing was therefore planned and Infectious Diseases consultation was obtained for appropriate preventions. The FDA Prescribing Information recommends Neisseria meningitidis vaccination before starting Eculizumab or, if immediate Eculizumab is necessary, to use antibiotic prophylaxis until 2 weeks after vaccination. The accepted protective titer after meningococcal vaccination is population based and uses the serum bactericidal assay (SBA). An antibody titer of >1:4 (human compliment) or 1:8 (rabbit complement) is considered protective. However, this “gold standard” assay incorporates the use of exogenous human or rabbit complement. The protective SBA titers in subjects with terminal complement component deficiencies may not be properly assessed using these same SBA titer protective thresholds. Furthermore, serious meningococcal infections have occurred after appropriate vaccination in patients receiving chronic Eculizumab treatments (ie for paroxysmal nocturnal hemoglobinuria). Finally, SBA protective levels after single Neisseria meningitidis vaccination have not been achieved in majorities of patients with renal failure receiving dialysis and or transplant immunosuppression. Conclusions The current Eculizumab prescribing information recommendations for vaccination and antimicrobial prophylaxis may be inadequate to prevent serious Neisseria infections. Repeated Neisseria meningitidis vaccination and extended antibiotic prophylaxis may afford better protection in patients chronically dosed with Eculizumab.

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Computation of condition-dependent proteome allocation reveals variability in the macro and micro nutrient requirements for growth

10.1101/2020.03.23.003236 ◽

2020 ◽

Cited By ~ 1

Author(s):

Colton J. Lloyd ◽

Jonathan Monk ◽

Laurence Yang ◽

Ali Ebrahim ◽

Bernhard O. Palsson

Keyword(s):

Amino Acids ◽

Growth Conditions ◽

Anaerobic Growth ◽

Metabolic Phenotype ◽

E Coli ◽

Scale Models ◽

Protein Components ◽

K 12 ◽

Genome Scale ◽

Prosthetic Groups

AbstractSustaining a robust metabolic network requires a balanced and fully functioning proteome. In addition to amino acids, many enzymes require cofactors (coenzymes and engrafted prosthetic groups) to function properly. Extensively validated genome-scale models of metabolism and gene expression (ME-models) have the unique ability to compute an optimal proteome composition underlying a metabolic phenotype, including the provision of all required cofactors. Here we use the ME-model for Escherichia coli K-12 MG1655 to computationally examine how environmental conditions change the proteome and its accompanying cofactor usage. We found that: (1) The cofactor requirements computed by the ME model mostly agree with the standard biomass objective function used in models of metabolism alone (M models); (2) ME-model computations reveal non-intuitive variability in cofactor use under different growth conditions; (3) An analysis of ME-model predicted protein use in aerobic and anaerobic conditions suggests an enrichment in the use of prebiotic amino acids in the proteins used to sustain anaerobic growth (4) The ME-model could describe how limitation in key protein components affect the metabolic state of E. coli. Genome-scale models have thus reached a level of sophistication where they reveal intricate properties of functional proteomes and how they support different E. coli lifestyles.

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Isolation of Two Kinds of E. coli K-12 Mutants for Lysophospholipase L2: One with an Elevated Level of the Enzyme and the Other Defective in It1

The Journal of Biochemistry ◽

10.1093/oxfordjournals.jbchem.a134805 ◽

1984 ◽

Vol 96 (1) ◽

pp. 137-145 ◽

Cited By ~ 12

Author(s):

Tetsuyuki KOBAYASHI ◽

Hirosh HOMMA ◽

Yumiko NATORI ◽

Ichiro KUDO ◽

Keizo INOUE ◽

...

Keyword(s):

Elevated Level ◽

The Other ◽

E Coli ◽

K 12

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ISOLATION AND CHARACTERIZATION OF RIBONUCLEIC ACID FROM RAT LENS

Canadian Journal of Biochemistry and Physiology ◽

10.1139/o62-007 ◽

1962 ◽

Vol 40 (1) ◽

pp. 41-47 ◽

Cited By ~ 1

Author(s):

Anima Devi

Keyword(s):

Amino Acids ◽

The Other ◽

Ocular Lens ◽

E Coli ◽

Isolation And Characterization ◽

Coiled Structure ◽

Calf Thymus ◽

Original Procedure ◽

Polynucleotide Chain

RNA from rat ocular lens has been isolated by a method based on Kirby's original procedure (7), but greatly modified so as to avoid any degradation of RNA by RNase during the process of its extraction from lenses. The absorption at 260 mμ of a 1.0% solution of this purified material in a 1-cm cell is 1.95. Its N/P ratio is 1.58. It has 20 to 25% activity of that of yeast-soluble RNA in accepting activated amino acids. When this RNA (like all other RNA's) is heated and cooled the polynucleotide chain can again form loops, thus suggesting a randomly coiled structure for this RNA. On the other hand, DNA preparations from calf thymus, rat liver, and E. coli showed irreversible changes when heated and cooled.

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The Bβ-sheet in the PAI-1 Molecule Plays an Important Role for Its Stability

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613940 ◽

2000 ◽

Vol 83 (06) ◽

pp. 896-901 ◽

Cited By ~ 4

Author(s):

Guang-Chao Sui ◽

Björn Wiman

Keyword(s):

Amino Acids ◽

Half Life ◽

Ph Dependence ◽

The Other ◽

Wild Type ◽

E Coli ◽

Reactive Center ◽

The Stability ◽

Β Sheet ◽

Pai 1

SummaryWe have investigated the B β-sheet in PAI-1 regarding its role for the stability of the molecule. The residues from His219 to Tyr241 (except for Gly230 and Pro240), covering the s2B and s3B strands, and in addition His185 and His190 were substituted by amino acids with opposite properties. The 23 generated single-site changed mutants and also wild type PAI-1 (wtPAI-1) were expressed in E. coli. Subsequently they were purified by heparin-Sepharose and anhydrotrypsin agarose affinity chromatographies. The stability of the purified PAI-1 variants was analyzed at 37° C and at different pHs (5.5, 6.5 or 7.5). At pH 7.5 and 37° C, single substitutions of the residues in the central portions of both strands 2 and 3 in the B β-sheet (Ile223 to Leu226 on s2B and Met235 to Ile237 on s3B), caused a significant decrease in stability, yielding half-lives of about 10–25% as compared to wtPAI-1. On the other hand, mutations at both sides of the central portion of the B β-sheet (Tyr221, Asp222, Tyr228 and Thr232) frequently resulted in an increased PAI-1 stability (up to 7-fold). While wtPAI-1 exhibited prolonged half-lives at pH 6.5 and 5.5, the PAI-1 variant Y228S was more stable at neutral pH (half-life of 9.6 h at pH 7.5) as compared to its half-life at pH 5.5 (1.1 h). One of the 4 modified histidine residues (His229) resulted in a variant with a clearly affected stability as a function of pH, suggesting that it may, at least in part, be of importance for the pH dependence of the PAI-1 stability. Thus, our data demonstrate that the B β-sheet is of great importance for the stability of the molecule. Modifications in this part causes decreased or increased stability in a certain pattern, suggesting effects on the insertion rate of the reactive center loop into the A β-sheet of the molecule.

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Efficacy of Human Monoclonal Antibodies to the Group B Polysaccharide of Neisseria Meningitidis in Experimental Infant Rat Model

Neisseriae 1990 ◽

10.1515/9783110867787-050 ◽

1991 ◽

pp. 271-276

Author(s):

K. Saukkonen ◽

M. Leinonen ◽

H.V. Raff

Keyword(s):

Monoclonal Antibodies ◽

Neisseria Meningitidis ◽

Rat Model ◽

Human Monoclonal Antibodies ◽

Infant Rat ◽

Group B

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Neisseria meningitidis group B serosubtyping using monoclonal antibodies in whole-cell Elisa

Microbial Pathogenesis ◽

10.1016/0882-4010(88)90045-9 ◽

1988 ◽

Vol 4 (1) ◽

pp. 27-32 ◽

Cited By ~ 68

Author(s):

Hussein Abdillahi ◽

Jan T. Poolman

Keyword(s):

Monoclonal Antibodies ◽

Neisseria Meningitidis ◽

Whole Cell ◽

Group B ◽

Cell Elisa

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Quorum-Sensing Escherichia coli Regulator A: a Regulator of the LysR Family Involved in the Regulation of the Locus of Enterocyte Effacement Pathogenicity Island in Enterohemorrhagic E. coli

Infection and Immunity ◽

10.1128/iai.70.6.3085-3093.2002 ◽

2002 ◽

Vol 70 (6) ◽

pp. 3085-3093 ◽

Cited By ~ 128

Author(s):

Vanessa Sperandio ◽

Caiyi C. Li ◽

James B. Kaper

Keyword(s):

Escherichia Coli ◽

Quorum Sensing ◽

Type Iii Secretion ◽

Pathogenicity Island ◽

The Other ◽

E Coli ◽

Type Iii ◽

Enterocyte Effacement ◽

K 12 ◽

Lysr Family

ABSTRACT The locus of enterocyte effacement (LEE) is a chromosomal pathogenicity island that encodes the proteins involved in the formation of the attaching and effacing lesions by enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). The LEE comprises 41 open reading frames organized in five major operons, LEE1, LEE2, LEE3, tir (LEE5), and LEE4, which encode a type III secretion system, the intimin adhesin, the translocated intimin receptor (Tir), and other effector proteins. The first gene of LEE1 encodes the Ler regulator, which activates all the other genes within the LEE. We previously reported that the LEE genes were activated by quorum sensing through Ler (V. Sperandio, J. L. Mellies, W. Nguyen, S. Shin, and J. B. Kaper, Proc. Natl. Acad. Sci. USA 96:15196-15201, 1999). In this study we report that a putative regulator in the E. coli genome is itself activated by quorum sensing. This regulator is encoded by open reading frame b3243; belongs to the LysR family of regulators; is present in EHEC, EPEC, and E. coli K-12; and shares homology with the AphB and PtxR regulators of Vibrio cholerae and Pseudomonas aeruginosa, respectively. We confirmed the activation of b3243 by quorum sensing by using transcriptional fusions and renamed this regulator quorum-sensing E. coli regulator A (QseA). We observed that QseA activated transcription of ler and therefore of the other LEE genes. An EHEC qseA mutant had a striking reduction of type III secretion activity, which was complemented when qseA was provided in trans. Similar results were also observed with a qseA mutant of EPEC. The QseA regulator is part of the regulatory cascade that regulates EHEC and EPEC virulence genes by quorum sensing.

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