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Berlin Brandenburg

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  • 1
    Language: English
    In: PloS one, 2015, Vol.10(4), pp.e0124373
    Description: Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. The triple mutant was significantly more susceptible to both α- and β-defensins; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin LL-37 may be more important than defensin resistance to H. ducreyi pathogenesis.
    Keywords: Bacterial Proteins -- Genetics ; Drug Resistance, Bacterial -- Genetics ; Ethanolaminephosphotransferase -- Genetics ; Haemophilus Ducreyi -- Genetics ; Lipid A -- Metabolism
    E-ISSN: 1932-6203
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  • 2
    Language: English
    In: PloS one, 2017, Vol.12(6), pp.e0179621
    Description: Neisseria gonorrhoeae, the causative agent of gonorrhea, has a number of factors known to contribute to pathogenesis; however, a full understanding of these processes and their regulation has proven to be elusive. Post-translational modifications (PTMs) of bacterial proteins are now recognized as one mechanism of protein regulation. In the present study, Western blot analyses, with an anti-acetyl-lysine antibody, indicated that a large number of gonococcal proteins are post-translationally modified. Previous work has shown that Nε-lysine acetylation can occur non-enzymatically with acetyl-phosphate (AcP) as the acetyl donor. In the current study, an acetate kinase mutant (1291ackA), which accumulates AcP, was generated in N. gonorrhoeae. Broth cultures of N. gonorrhoeae 1291wt and 1291ackA were grown, proteins extracted and digested, and peptides containing acetylated-lysines (K-acetyl) were affinity-enriched from both strains. Mass spectrometric analyses of these samples identified a total of 2686 unique acetylation sites. Label-free relative quantitation of the K-acetyl peptides derived from the ackA and wild-type (wt) strains demonstrated that 109 acetylation sites had an ackA/wt ratio〉2 and p-values 〈0.05 in at least 2/3 of the biological replicates and were designated as "AckA-dependent". Regulated K-acetyl sites were found in ribosomal proteins, central metabolism proteins, iron acquisition and regulation proteins, pilus assembly and regulation proteins, and a two-component response regulator. Since AckA is part of a metabolic pathway, comparative growth studies of the ackA mutant and wt strains were performed. The mutant showed a growth defect under aerobic conditions, an inability to grow anaerobically, and a defect in biofilm maturation. In conclusion, the current study identified AckA-dependent acetylation sites in N. gonorrhoeae and determined that these sites are found in a diverse group of proteins. This work lays the foundation for future studies focusing on specific acetylation sites that may have relevance in gonococcal pathogenesis and metabolism.
    Keywords: Acetate Kinase -- Metabolism ; Bacterial Proteins -- Metabolism ; Metabolic Networks and Pathways -- Physiology ; Neisseria Gonorrhoeae -- Metabolism
    E-ISSN: 1932-6203
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  • 3
    Language: English
    In: PLoS ONE, 2012, Vol.7(6), p.e38303
    Description: Neisseria gonorrhoeae , the causative agent of gonorrhea, can form biofilms in vitro and in vivo . In biofilms, the organism is more resistant to antibiotic treatment and can serve as a reservoir for chronic infection. We have used stable isotope labeling by amino acids in cell culture (SILAC) to compare protein expression in biofilm and planktonic organisms. Two parallel populations of N. gonorrhoeae strain 1291, which is an arginine auxotroph, were grown for 48 h in continuous-flow chambers over glass, one supplemented with 13 C 6 -arginine for planktonic organisms and the other with unlabeled arginine for biofilm growth. The biofilm and planktonic cells were harvested and lysed separately, and fractionated into three sequential protein extracts. Corresponding heavy (H) planktonic and light (L) biofilm protein extracts were mixed and separated by 1D SDS-PAGE gels, and samples were extensively analyzed by liquid chromatography-mass spectrometry. Overall, 757 proteins were identified, and 152 unique proteins met a 1.5-fold cutoff threshold for differential expression with p-values 〈0.05. Comparing biofilm to planktonic organisms, this set included 73 upregulated and 54 downregulated proteins. Nearly a third of the upregulated proteins were involved in energy metabolism, with cell envelope proteins making up the next largest group. Of the downregulated proteins, the largest groups were involved in protein synthesis and energy metabolism. These proteomics results were compared with our previously reported results from transcriptional profiling of gonococcal biofilms using microarrays. Nitrite reductase and cytochrome c peroxidase, key enzymes required for anaerobic growth, were detected as highly upregulated in both the proteomic and transcriptomic datasets. These and other protein expression changes observed in the present study were consistent with a shift to anaerobic respiration in gonococcal biofilms, although changes in membrane proteins not explicitly related to this shift may have other functions.
    Keywords: Research Article ; Biology ; Microbiology ; Biochemistry
    E-ISSN: 1932-6203
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  • 4
    Language: English
    In: PLoS ONE, 2010, Vol.5(7), p.e11060
    Description: Capsular polysaccharides are important factors in bacterial pathogenesis and have been the target of a number of successful vaccines. Francisella tularensis has been considered to express a capsular antigen but none has been isolated or characterized. We have developed a monoclonal antibody, 11B7, which recognizes the capsular polysaccharide of F. tularensis migrating on Western blot as a diffuse band between 100 kDa and 250 kDa. The capsule stains poorly on SDS-PAGE with silver stain but can be visualized using ProQ Emerald glycoprotein stain. The capsule appears to be highly conserved among strains of F. tularensis as antibody 11B7 bound to the capsule of 14 of 14 F. tularensis type A and B strains on Western blot. The capsular material can be isolated essentially free of LPS, is phenol and proteinase K resistant, ethanol precipitable and does not dissociate in sodium dodecyl sulfate. Immunoelectron microscopy with colloidal gold demonstrates 11B7 circumferentially staining the surface of F. tularensis which is typical of a polysaccharide capsule. Mass spectrometry, compositional analysis and NMR indicate that the capsule is composed of a polymer of the tetrasaccharide repeat, 4)-α-D-GalNAcAN-(1-〉4)-α-D-GalNAcAN-(1-〉3)-β-D-QuiNAc-(1-〉2)-β-D-Qui4NFm-(1-, which is identical to the previously described F. tularensis O-antigen subunit. This indicates that the F. tularensis capsule can be classified as an O-antigen capsular polysaccharide. Our studies indicate that F. tularensis O-antigen glycosyltransferase mutants do not make a capsule. An F. tularensis acyltransferase and an O-antigen polymerase mutant had no evidence of an O-antigen but expressed a capsular antigen. Passive immunization of BALB/c mice with 75 µg of 11B7 protected against a 150 fold lethal challenge of F. tularensis LVS. Active immunization of BALB/c mice with 10 µg of capsule showed a similar level of protection. These studies demonstrate that F. tularensis produces an O-antigen capsule that may be the basis of a future vaccine.
    Keywords: Research Article ; Microbiology ; Microbiology -- Cellular Microbiology And Pathogenesis ; Microbiology -- Immunity To Infections ; Microbiology -- Medical Microbiology
    E-ISSN: 1932-6203
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  • 5
    In: mBio, 2018, Vol.9(4)
    Description: ABSTRACT Nontypeable Haemophilus influenzae (NTHi) is an important pathogen in individuals of all ages. The lipooligosaccharide (LOS) of NTHi has evolved a complex structure that can be attributed to a multiplicity of glycosyltransferases, the random switching of glycosyltransferase gene expression via phase variation, and the complex structure of its core region with multiple glycoform branch points. This article adds to that complexity by describing a multifunctional enzyme (LsgB) which optimally functions when the species is grown on a solid surface and which can add either a ketodeoxyoctanoate (KDO) or an N -acetylneuramic acid (Neu5Ac) moiety to a terminal N- acetyllactosamine structure of LOS. Our studies show that expression of lsgB is reduced four- to sixfold when NTHi is grown in broth. The substrate that the enzyme utilizes is dependent upon the concentration of free Neu5Ac (between 1 and 10 µg/ml) in the environment. In environments in which Neu5Ac is below that level, the enzyme utilizes endogenous CMP-KDO as the substrate. Our studies show that during in vivo growth in an NTHi biofilm, the KDO moiety is expressed by the organism. Monoclonal antibody 6E4, which binds KDO, is bactericidal for NTHi strains that express the KDO epitope at high levels. In a survey of 33 NTHi strains isolated from healthy and diseased individuals, the antibody was bactericidal (〉90% kill) for 12 strains (36%). These studies open up the possibility of using a KDO-based glycoconjugate vaccine as part of a multicomponent vaccine against NTHi. IMPORTANCE Nontypeable Haemophilus influenzae is an important pathogen in middle ear infections in children, sinusitis in adults, and acute bronchitis in individuals with chronic obstructive lung disease. The organism is very well adapted to the human host environment, and this has hindered successful development of an effective vaccine. In this article, we describe a mechanism by which the bacteria decorates its surface lipooligosaccharide with a sugar unique to Gram-negative bacteria, ketodeoxyoctanoate (KDO). This sugar decoration is present during active infection and we have shown that an antibody directed against this sugar can result in killing of the organism. These data demonstrate that the lipooligosaccharide ketodeoxyoctanoate epitope may be a novel NTHi - specific candidate vaccine antigen.
    Keywords: Research Article ; Biofilm ; Elisa ; Lipooligosaccharide ; Bactericidal Activity ; Keto-Deoxyoctanoate ; Nontypeable ; Sialic Acid ; Vaccine
    E-ISSN: 2150-7511
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  • 6
    Language: English
    In: mBio, 10 October 2017, Vol.8(5)
    Description: is the causative agent of tularemia and a potential bioterrorism agent. In the present study, we isolated, identified, and quantified the proteins present in the membranes of the virulent type A strain, Schu S4, and the attenuated type B strain, LVS (live vaccine strain). Spectral counting of mass spectrometric data showed enrichment for membrane proteins in both strains. Mice vaccinated with whole LVS membranes encapsulated in poly (lactic-co-glycolic acid) (PLGA) nanoparticles containing the adjuvant polyinosinic-polycytidylic acid [poly(I·C)] showed significant protection against a challenge with LVS compared to the results seen with naive mice or mice vaccinated with either membranes or poly(I·C) alone. The PLGA-encapsulated Schu S4 membranes with poly(I·C) alone did not significantly protect mice from a lethal intraperitoneal challenge with Schu S4; however, this vaccination strategy provided protection from LVS challenge. Mice that received the encapsulated Schu S4 membranes followed by a booster of LVS bacteria showed significant protection with respect to a lethal Schu S4 challenge compared to control mice. Western blot analyses of the sera from the Schu S4-vaccinated mice that received an LVS booster showed four immunoreactive bands. One of these bands from the corresponding one-dimensional (1D) SDS-PAGE experiment represented capsule. The remaining bands were excised, digested with trypsin, and analyzed using mass spectrometry. The most abundant proteins present in these immunoreactive samples were an outer membrane OmpA-like protein, FopA; the type IV pilus fiber building block protein; a hypothetical membrane protein; and lipoproteins LpnA and Lpp3. These proteins should serve as potential targets for future recombinant protein vaccination studies. The low infectious dose, the high potential mortality/morbidity rates, and the ability to be disseminated as an aerosol make a potential agent for bioterrorism. These characteristics led the Centers for Disease Control (CDC) to classify as a Tier 1 pathogen. Currently, there is no vaccine approved for general use in the United States.
    Keywords: Franscisella Tularensis ; Immunity ; Inner Membrane Proteins ; Outer Membrane Proteins ; Proteomics ; Bacterial Vaccines -- Immunology ; Francisella Tularensis -- Immunology ; Membrane Proteins -- Immunology ; Tularemia -- Prevention & Control ; Vaccines, Subunit -- Immunology
    E-ISSN: 2150-7511
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  • 7
    Language: English
    In: Infection and immunity, 04 January 2016, Vol.84(3), pp.765-74
    Description: Haemophilus haemolyticus and nontypeable Haemophilus influenzae (NTHi) are closely related upper airway commensal bacteria that are difficult to distinguish phenotypically. NTHi causes upper and lower airway tract infections in individuals with compromised airways, while H. haemolyticus rarely causes such infections. The lipooligosaccharide (LOS) is an outer membrane component of both species and plays a role in NTHi pathogenesis. In this study, comparative analyses of the LOS structures and corresponding biosynthesis genes were performed. Mass spectrometric and immunochemical analyses showed that NTHi LOS contained terminal sialic acid more frequently and to a higher extent than H. haemolyticus LOS did. Genomic analyses of 10 strains demonstrated that H. haemolyticus lacked the sialyltransferase genes lic3A and lic3B (9/10) and siaA (10/10), but all strains contained the sialic acid uptake genes siaP and siaT (10/10). However, isothermal titration calorimetry analyses of SiaP from two H. haemolyticus strains showed a 3.4- to 7.3-fold lower affinity for sialic acid compared to that of NTHi SiaP. Additionally, mass spectrometric and immunochemical analyses showed that the LOS from H. haemolyticus contained phosphorylcholine (ChoP) less frequently than the LOS from NTHi strains. These differences observed in the levels of sialic acid and ChoP incorporation in the LOS structures from H. haemolyticus and NTHi may explain some of the differences in their propensities to cause disease.
    Keywords: Haemophilus -- Metabolism ; Haemophilus Infections -- Microbiology ; Haemophilus Influenzae -- Metabolism ; Lipopolysaccharides -- Chemistry ; N-Acetylneuraminic Acid -- Analysis ; Phosphorylcholine -- Analysis
    ISSN: 00199567
    E-ISSN: 1098-5522
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  • 8
    Language: English
    In: Infection and immunity, April 2014, Vol.82(4), pp.1523-39
    Description: The virulence factors mediating Francisella pathogenesis are being investigated, with an emphasis on understanding how the organism evades innate immunity mechanisms. Francisella tularensis produces a lipopolysaccharide (LPS) that is essentially inert and a polysaccharide capsule that helps the organism to evade detection by components of innate immunity. Using an F. tularensis Schu S4 mutant library, we identified strains that are disrupted for capsule and O-antigen production. These serum-sensitive strains lack both capsule production and O-antigen laddering. Analysis of the predicted protein sequences for the disrupted genes (FTT1236 and FTT1238c) revealed similarity to those for waa (rfa) biosynthetic genes in other bacteria. Mass spectrometry further revealed that these proteins are involved in LPS core sugar biosynthesis and the ligation of O antigen to the LPS core sugars. The 50% lethal dose (LD50) values of these strains are increased 100- to 1,000-fold for mice. Histopathology revealed that the immune response to the F. tularensis mutant strains was significantly different from that observed with wild-type-infected mice. The lung tissue from mutant-infected mice had widespread necrotic debris, but the spleens lacked necrosis and displayed neutrophilia. In contrast, the lungs of wild-type-infected mice had nominal necrosis, but the spleens had widespread necrosis. These data indicate that murine death caused by wild-type strains occurs by a mechanism different from that by which the mutant strains kill mice. Mice immunized with these mutant strains displayed 〉10-fold protective effects against virulent type A F. tularensis challenge.
    Keywords: Francisella Tularensis -- Pathogenicity ; O Antigens -- Genetics ; Tularemia -- Microbiology
    ISSN: 00199567
    E-ISSN: 1098-5522
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  • 9
    In: Molecular Microbiology, December 2018, Vol.110(5), pp.677-688
    Description: Post‐translational acetylation is a common protein modification in bacteria. It was recently reported that acetylates the Type IV pilus retraction motor, PilT. Here, we show recombinant PilT can be acetylated and acetylation does not affect PilT ultrastructure. To investigate the function of PilT acetylation, we mutated an acetylated lysine, K117, to mimic its acetylated or unacetylated forms. These mutations were not tolerated by wild‐type but they were tolerated by carrying an inducible when grown without inducer. We identified additional mutations in and that suppress the lethality of K117 mutations. To investigate the link between PilE and PilT acetylation, we found the lack of PilE decreases PilT acetylation levels and increases the amount of PilT associated with the inner membrane. Finally, we found no difference between wild‐type and mutant cells in transformation efficiency, suggesting neither mutation inhibits Type IV pilus retraction. Mutant cells, however, form microcolonies morphologically distinct from wt cells. We conclude that interfering with the acetylation status of PilT greatly reduces viability, and mutations in and can overcome this lethality. We discuss the implications of these findings in the context of Type IV pilus retraction regulation. Cells can control protein activity through post‐translational modification. Here, we show by genetic analysis that the viability of pathogen is dramatically reduced when its PilT protein is prevented from switching between acetylated and deacetylated states. Loss of viability is overcome when secondary mutations occur in genes encoding proteins known or suspected to interact with PilT at the membrane. These findings demonstrate that acetylation is an important determinant of PilT function, and imply that PilT influences membrane dynamics.
    Keywords: Lysine;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 10
    In: Infection and Immunity, 2002, Vol. 70(10), p.5808
    Description: The primary human urethral epithelial cells developed by our laboratory have been immortalized by transduction with a retroviral vector expressing the human papillomavirus E6E7 oncogenes. Analysis of telomerase expression and comparison to that in primary cells revealed detectable levels in the transduced human urethral epithelial cells. Immortalized urethral cells could be passaged over 20 times. Immunofluorescence microscopy studies showed that the immortalized cells were phenotypically similar and responded to gonococcal infection similarly to primary cells. Specifically, positive cytokeratin staining showed that the immortalized cells are keratinocytes; cell surface levels of human asialoglycoprotein receptor increase following gonococcal infection, and, like the primary cells, the immortalized urethral epithelial cells are CD14 negative. Using enzyme-linked immunosorbent assay, we found that interleukin-6 (IL-6) and IL-8 levels in primary urethral epithelial cell supernatants increase after challenge with N. gonorrhoeae. Likewise, the immortalized urethral epithelial cells produced higher levels of IL-6 and IL-8 cytokines in response to gonococcal infection. Cells challenged with a gonococcal lipid A msbB mutant produced reduced IL-6 and IL-8 levels when compared to the parent strain. Additionally, these data suggest that the 1291 msbB lipooligosaccharide may suppress cytokine induction.
    Keywords: Medicine ; Biology;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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