Susan M. Twine

A Mutant of Francisella tularensis Strain SCHU S4 Lacking the Ability To Express a 58-Kilodalton Protein Is Attenuated for Virulence and Is an Effective Live Vaccine

ABSTRACT Francisella tularensis subsp. tularensis (type A) strain SCHU S4 is a prototypic strain of the pathogen that is highly virulent for humans and other mammals. Its intradermal (i.d.) 50% lethal dose (LD50) for mice is <10 CFU. We discovered a spontaneous mutant, designated FSC043, of SCHU S4 with an i.d. LD50 of >108 CFU. FSC043 effectively vaccinated mice against challenge with a highly virulent type A strain, and the protective efficacy was at least as good as that of F. tularensis LVS, an empirically attenuated strain which has been used as an efficacious human vaccine. Comparative proteomics was used to identify two proteins of unknown function that were identified as defective in LVS and FSC043, and deletion mutants of SCHU S4 were created for each of the two encoding genes. One mutant, the ΔFTT0918 strain, failed to express a 58-kDa protein, had an i.d. LD50 of ∼105 CFU, and was found to be less capable than SCHU S4 of growing in peritoneal mouse macrophages. Mice that recovered from sublethal infection with the ΔFTT0918 mutant survived when challenged 2 months later with >100 LD50s of the highly virulent type A strain FSC033. This is the first report of the generation of defined mutants of F. tularensis subsp. tularensis and their use as live vaccines.

Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum

BackgroundProteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray.ResultsWhole genome microarray analysis revealed that 63% of the coding sequences (CDSs) present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes), and the flagellar glycosylation island (FGI). These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5) has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI.ConclusionProteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism outbreaks.

Motility and Flagellar Glycosylation in Clostridium difficile

In this study, intact flagellin proteins were purified from strains of Clostridium difficile and analyzed using quadrupole time of flight and linear ion trap mass spectrometers. Top-down studies showed the flagellin proteins to have a mass greater than that predicted from the corresponding gene sequence. These top-down studies revealed marker ions characteristic of glycan modifications. Additionally, diversity in the observed masses of glycan modifications was seen between strains. Electron transfer dissociation mass spectrometry was used to demonstrate that the glycan was attached to the flagellin protein backbone in O linkage via a HexNAc residue in all strains examined. Bioinformatic analysis of C. difficile genomes revealed diversity with respect to glycan biosynthesis gene content within the flagellar biosynthesis locus, likely reflected by the observed flagellar glycan diversity. In C. difficile strain 630, insertional inactivation of a glycosyltransferase gene (CD0240) present in all sequenced genomes resulted in an inability to produce flagellar filaments at the cell surface and only minor amounts of unmodified flagellin protein.

Flagellar glycosylation in Clostridium botulinum

Flagellins from Clostridium botulinum were shown to be post‐translationally modified with novel glycan moieties by top‐down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O‐linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of legionaminic acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel legionaminic acid derivative, 7‐acetamido‐5‐(N‐methyl‐glutam‐4‐yl)‐amino‐3,5,7,9‐tetradeoxy‐d‐glycero‐α‐d‐galacto‐nonulosonic acid, (αLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision‐activated dissociation was utilized to map seven sites of O‐linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C‐terminal flagellin peptide marker ion, were identified in a top‐down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram‐positive flagellar proteins by the ‘sialic acid‐like’ nonulosonate sugar, legionaminic acid.

Modulation of Toxin Production by the Flagellar Regulon in Clostridium difficile

ABSTRACT We show in this study that toxin production in Clostridium difficile is altered in cells which can no longer form flagellar filaments. The impact of inactivation of fliC, CD0240, fliF, fliG, fliM, and flhB-fliR flagellar genes upon toxin levels in culture supernatants was assessed using cell-based cytotoxicity assay, proteomics, immunoassay, and immunoblotting approaches. Each of these showed that toxin levels in supernatants were significantly increased in a fliC mutant compared to that in the C. difficile 630 parent strain. In contrast, the toxin levels in supernatants secreted from other flagellar mutants were significantly reduced compared with that in the parental C. difficile 630 strain. Transcriptional analysis of the pathogenicity locus genes (tcdR, tcdB, tcdE, and tcdA) revealed a significant increase of all four genes in the fliC mutant strain, while transcription of all four genes was significantly reduced in fliM, fliF, fliG, and flhB-fliR mutants. These results demonstrate that toxin transcription in C. difficile is modulated by the flagellar regulon. More significantly, mutant strains showed a corresponding change in virulence compared to the 630 parent strain when tested in a hamster model of C. difficile infection. This is the first demonstration of differential flagellum-related transcriptional regulation of toxin production in C. difficile and provides evidence for elaborate regulatory networks for virulence genes in C. difficile.

Flagellar glycosylation in Burkholderia pseudomallei and Burkholderia thailandensis.

Glycosylation of proteins is known to impart novel physical properties and biological roles to proteins from both eukaryotes and prokaryotes. In this study, gel-based glycoproteomics were used to identify glycoproteins of the potential biothreat agent Burkholderia pseudomallei and the closely related but nonpathogenic B. thailandensis. Top-down and bottom-up mass spectrometry (MS) analyses identified that the flagellin proteins of both species were posttranslationally modified by novel glycans. Analysis of proteins from two strains of each species demonstrated that B. pseudomallei flagellin proteins were modified with a glycan with a mass of 291 Da, while B. thailandensis flagellin protein was modified with related glycans with a mass of 300 or 342 Da. Structural characterization of the B. thailandensis carbohydrate moiety suggests that it is an acetylated hexuronic acid. In addition, we have identified through mutagenesis a gene from the lipopolysaccharide (LPS) O-antigen biosynthetic cluster which is involved in flagellar glycosylation, and inactivation of this gene eliminates flagellar glycosylation and motility in B. pseudomallei. This is the first report to conclusively demonstrate the presence of a carbohydrate covalently linked to a protein in B. pseudomallei and B. thailandensis, and it suggests new avenues to explore in order to examine the marked differences in virulence between these two species.

Flagellin Diversity in Clostridium botulinum Groups I and II: a New Strategy for Strain Identification

ABSTRACT Strains of Clostridium botulinum are traditionally identified by botulinum neurotoxin type; however, identification of an additional target for typing would improve differentiation. Isolation of flagellar filaments and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that C. botulinum produced multiple flagellin proteins. Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis of in-gel tryptic digests identified peptides in all flagellin bands that matched two homologous tandem flagellin genes identified in the C. botulinum Hall A genome. Designated flaA1 and flaA2, these open reading frames encode the major structural flagellins of C. botulinum. Colony PCR and sequencing of flaA1/A2 variable regions classified 80 environmental and clinical strains into group I or group II and clustered isolates into 12 flagellar types. Flagellar type was distinct from neurotoxin type, and epidemiologically related isolates clustered together. Sequencing a larger PCR product, obtained during amplification of flaA1/A2 from type E strain Bennett identified a second flagellin gene, flaB. LC-MS analysis confirmed that flaB encoded a large type E-specific flagellin protein, and the predicted molecular mass for FlaB matched that observed by SDS-PAGE. In contrast, the molecular mass of FlaA was 2 to 12 kDa larger than the mass predicted by the flaA1/A2 sequence of a given strain, suggesting that FlaA is posttranslationally modified. While identification of FlaB, and the observation by SDS-PAGE of different masses of the FlaA proteins, showed the flagellin proteins of C. botulinum to be diverse, the presence of the flaA1/A2 gene in all strains examined facilitates single locus sequence typing of C. botulinum using the flagellin variable region.

BALB/c mice, but not C57BL/6 mice immunized with a ΔclpB mutant of Francisella tularensis subspecies tularensis are protected against respiratory challenge with wild-type bacteria: Association of protection with post-vaccination and post-challenge immune responses

Francisella tularensis subspecies tularensis is highly virulent for humans especially when it is inhaled. Therefore, it has the potential to be used as a biothreat agent. Vaccines against F. tularensis will need to be approved in accordance with the FDA Animal Rule. This will require identification of robust correlates of protection in experimental animals and the demonstration that similar immune responses are generated in vaccinated humans. Towards this goal, we have developed an experimental live vaccine strain by deleting the gene, clpB, encoding a heat shock protein from virulent subsp. tularensis strain, SCHU S4. SCHU S4ΔclpB administered intradermally protects BALB/c, but not C57BL/6 mice from subsequent respiratory challenge with wildtype SCHU S4. A comparison of post-vaccination and post-challenge immune responses in these two mouse strains shows an association between several antibody and cytokine responses and protection. In particular, elevated IFNγ levels in the skin 2 days after vaccination, sero-conversion to hypothetical membrane protein FTT_1778c, and to 30S ribosomal protein S1 (FTT_0183c) of F. tularensis after 30 days of vaccination, and elevated levels of pulmonary IL-17 on day 7 after respiratory challenge with SCHU S4 were all associated with protection.

Proteomic insights into cold adaptation of psychrotrophic and mesophilic Acidithiobacillus ferrooxidans strains

Cold tolerant strains of Acidithiobacillus ferrooxidans play a role in metal leaching and acid mine drainage (AMD) production in northern latitude/boreal mining environments. In this study we used a proteomics and bioinformatics approach to decipher the proteome changes related to sustained growth at low temperatures to increase our understanding of cold adaptation mechanisms in A. ferrooxidans strains. Changes in protein abundance in response to low temperatures (5 and 15°C) were monitored and protein analyses of a psychrotrophic strain (D6) versus a mesophilic strain (F1) showed that both strains increased levels of 11 stress-related and metabolic proteins including survival protein SurA, trigger factor Tig, and AhpC-Tsa antioxidant proteins. However, a unique set of changes in the proteome of psychrotrophic strain D6 were observed. In particular, the importance of protein fate, membrane transport and structure for psychrotrophic growth were evident with increases in numerous chaperone and transport proteins including GroEL, SecB, ABC transporters and a capsule polysaccharide export protein. We also observed that low temperature iron oxidation coincides with a relative increase in the key iron metabolism protein rusticyanin, which was more highly expressed in strain D6 than in strain F1 at colder growth temperatures. We demonstrate that the psychrotrophic strain uses a global stress response and cold-active metabolism which permit growth of A. ferrooxidans in the extreme AMD environment in colder climates.

Differential Glycosylation of Polar and Lateral Flagellins in Aeromonas hydrophila AH-3

Background: Aeromonas hydrophila flagellar glycosylation is crucial for flagellar production. Results: Aeromonas hydrophila strain AH-3 lateral and polar flagellins are modified in O-linkage with a single monosaccharide or a heterogenous glycan (heptasaccharide), respectively. Mutants in pseudaminic biosynthesis abolish flagellar biogenesis and flagellins expression. Conclusion: Flagellar formation depends on glycosylation. Significance: O-Glycosylation by two different glycans for polar and lateral flagellins and is crucial for flagellar biogenesis. Polar and lateral flagellin proteins from Aeromonas hydrophila strain AH-3 (serotype O34) were found to be glycosylated with different carbohydrate moieties. The lateral flagellin was modified at three sites in O-linkage, with a single monosaccharide of 376 Da, which we show to be a pseudaminic acid derivative. The polar flagellin was modified with a heterogeneous glycan, comprised of a heptasaccharide, linked through the same 376-Da sugar to the protein backbone, also in O-linkage. In-frame deletion mutants of pseudaminic acid biosynthetic genes pseB and pseF homologues resulted in abolition of polar and lateral flagellar formation by posttranscriptional regulation of the flagellins, which was restored by complementation with wild type pseB or F homologues or Campylobacter pseB and F.