Analysis of the cell surface layer ultrastructure of the oral pathogen Tannerella forsythia

The Gram-negative oral pathogen Tannerella forsythia is decorated with a 2D crystalline surface (S-) layer, with two different S-layer glycoprotein species being present. Prompted by the predicted virulence potential of the S-layer, this study focused on the analysis of the arrangement of the individual S-layer glycoproteins by a combination of microscopic, genetic, and biochemical analyses. The two S-layer genes are transcribed into mRNA and expressed into protein in equal amounts. The S-layer was investigated on intact bacterial cells by transmission electron microscopy, by immune fluorescence microscopy, and by atomic force microscopy. The analyses of wild-type cells revealed a distinct square S-layer lattice with an overall lattice constant of 10.1 ± 0.7 nm. In contrast, a blurred lattice with a lattice constant of 9.0 nm was found on S-layer single-mutant cells. This together with in vitro self-assembly studies using purified (glyco)protein species indicated their increased structural flexibility after self-assembly and/or impaired self-assembly capability. In conjunction with TEM analyses of thin-sectioned cells, this study demonstrates the unusual case that two S-layer glycoproteins are co-assembled into a single S-layer. Additionally, flagella and pilus-like structures were observed on T. forsythia cells, which might impact the pathogenicity of this bacterium

Rugged Single Domain Antibody Detection Elements for Bacillus anthracis Spores and Vegetative Cells

Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs) were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors

Crucial residues in the carboxy-terminal end of C1 inhibitor revealed by pathogenic mutants impaired in secretion or function.

The last exon of the C1-1NH gene was screened for point mutations in 36 unrelated hereditary angioedema patients. Mutations were found in eight patients, predicting changes in the short COOH-terminal region which anchors the reactive site loop on its COOH-terminal side. The effects of each of these mutations were examined in transiently transfected Cos-7 cells. Complete intracellular retention or degradation was observed with substitutions in the COOH-terminal strands 4B or 5B: Leu459-->Pro, Leu459-->Arg, and Pro467-->Arg were all blocked at early stages of intracellular transport, but differences in the immunofluorescence patterns indicated that a significant fraction of the Leu459-->Pro and of the Pro467-->Arg proteins reached a compartment distinct from the endoplasmic reticulum. In line with previous findings with alpha 1-antitrypsin, chain termination within strand 5B resulted in rapid degradation. Mutant Val451-->Met, in strand 1C, and mutant Pro476-->Ser, replacing the invariant proline near the COOH terminus, yielded reduced secretion, but these extracellular proteins were unable to bind the target protease C1s. Presence of low levels of both dysfunctional proteins in patient plasmas defies the conventional classification of C1 inhibitor deficiencies as type I or type II. These data point to a key role of certain residues in the conserved COOH-terminal region of serpins in determining the protein foldings compatible with transport and proper exposure of the reactive site loop

Capsules, Toxins and AtxA as Virulence Factors of Emerging Bacillus cereus Biovar anthracis

Emerging B. cereus strains that cause anthrax-like disease have been isolated in Cameroon (CA strain) and Côte d’Ivoire (CI strain). These strains are unusual, because their genomic characterisation shows that they belong to the B. cereus species, although they harbour two plasmids, pBCXO1 and pBCXO2, that are highly similar to the pXO1 and pXO2 plasmids of B. anthracis that encode the toxins and the polyglutamate capsule respectively. The virulence factors implicated in the pathogenicity of these B. cereus bv anthracis strains remain to be characterised. We tested their virulence by cutaneous and intranasal delivery in mice and guinea pigs; they were as virulent as wild-type B. anthracis. Unlike as described for pXO2-cured B. anthracis, the CA strain cured of the pBCXO2 plasmid was still highly virulent, showing the existence of other virulence factors. Indeed, these strains concomitantly expressed a hyaluronic acid (HA) capsule and the B. anthracis polyglutamate (PDGA) capsule. The HA capsule was encoded by the hasACB operon on pBCXO1, and its expression was regulated by the global transcription regulator AtxA, which controls anthrax toxins and PDGA capsule in B. anthracis. Thus, the HA and PDGA capsules and toxins were co-regulated by AtxA. We explored the respective effect of the virulence factors on colonisation and dissemination of CA within its host by constructing bioluminescent mutants. Expression of the HA capsule by itself led to local multiplication and, during intranasal infection, to local dissemination to the adjacent brain tissue. Co-expression of either toxins or PDGA capsule with HA capsule enabled systemic dissemination, thus providing a clear evolutionary advantage. Protection against infection by B. cereus bv anthracis required the same vaccination formulation as that used against B. anthracis. Thus, these strains, at the frontier between B. anthracis and B. cereus, provide insight into how the monomorphic B. anthracis may have emerged

Structure and assembly of a Clostridioides difficile spore polar appendage

Clostridioides difficile, a strict anaerobic spore-former, is the main cause of nosocomial disease associated to antibiotic therapy in adults and a growing concern in the community. Spores are the main infectious, persistence and transmission vehicle. Spore germination occurs in the intestine and the resulting vegetative cells will produce the toxins responsible for the disease symptoms, and spores. During sporulation, a wild type population bifurcates into two main spore morphotypes, with or without a thick exosporium. We show that this bifurcation extends to the formation of spores with a robust polar appendage or spores with a short appendage or that lack this structure. The cysteine-rich CdeM protein localizes to the appendage and around the entire surface of the spore, and is a major structural component of the exosporium, which we show is continuous with the appendage. In a CdeM mutant, when present, the polar appendage is short and disorganized. We show that wild type and cdeM spores with a short or no appendage germinate poorly in response to taurocholate, compared to those with an appendage. cdeM spores of the two types, however, germinate faster than their wild type counterparts. Thus, while the absence of CdeM may increase the permeability of spores to taurocholate, proper assembly of the appendage is also important for germination. Consistent with an overall enhancement of germination, a cdeM mutant shows increased virulence in a hamster model of disease. For a wild type population, spores with a short or no appendage germinate slower than the appendage-bearing spores. Differences in transmission, persistence and disease severity may result, in part, from their proportion in a spore population.<br/

The <i>B</i>. <i>cereus</i> bv <i>anthracis</i> CA strain expresses a PDGA and a HA capsule, and toxins.

<p><b>(A)</b> Capsule expression in the CAP(Δ<i>pagA</i>), the CAR and the CAR-H(Δ<i>hasA</i>) strains in inducing conditions; the polyglutamate (PDGA) and hyaluronic acid (HA) capsule was visualised by immunofluorescence with a polyclonal anti-PDGA immune serum or by India ink staining; degradation of the HA capsule was achieved by incubation with hyaluronidase as described in the Materials and Methods section. (<b>B)</b> The production of toxin components PA and LF in overnight bacterial culture supernatants was determined by western blot with or without CO₂/bicarbonate as described in the Materials and Methods section.</p

Virulence of the <i>Bacillus cereus</i> bv <i>anthracis</i> and CA derivative strains by the subcutaneous route in guinea pigs.

<p>Guinea pigs were inoculated with graded spore inocula of each strain by subcutaneous route in the flank (four animals per dose). The presence of pBCXO1 and PBCXO2 and the gene inactivated on pBCXO1 is specified where applicable. Results are expressed as mean lethal dose (LD50) and mean time to death in days (MTD, mean ± SD). Each experiment was performed at least twice.</p><p>Virulence of the <i>Bacillus cereus</i> bv <i>anthracis</i> and CA derivative strains by the subcutaneous route in guinea pigs.</p