Proteolytic cleavage of pertussis toxin S1 subunit is not essential for its activity in mammalian cells

BACKGROUND: Pertussis toxin (PT) is an exotoxin virulence factor produced by Bordetella pertussis, the causative agent of whooping cough. PT consists of an active subunit (S1) that ADP-ribosylates the alpha subunit of several mammalian G proteins, and a B oligomer (S2–S5) that binds glycoconjugate receptors on cells. PT appears to enter cells by endocytosis, and retrograde transport through the Golgi apparatus may be important for its cytotoxicity. A previous study demonstrated that proteolytic processing of S1 occurs after PT enters mammalian cells. We sought to determine whether this proteolytic processing of S1 is necessary for PT cytotoxicity. RESULTS: Protease inhibitor studies suggested that S1 processing may involve a metalloprotease, and processing does not involve furin, a mammalian cell protease that cleaves several other bacterial toxins. However, inhibitor studies showed a general lack of correlation of S1 processing with PT cellular activity. A combination of replacement, insertion and deletion mutations in the C-terminal region of S1, as well as mass spectrometry data, suggested that the cleavage site is located around residue 203–204, but that cleavage is not strongly sequence-dependent. Processing of S1 was abolished by each of 3 overlapping 8 residue deletions just downstream of the putative cleavage site, but not by smaller deletions in the same region. Processing of the various mutant forms of PT did not correlate with cellular activity of the toxin, nor with the ability of the bacteria producing them to infect the mouse respiratory tract. In addition, S1 processing was not detected in transfected cells expressing S1, even though S1 was fully active in these cells. CONCLUSIONS: S1 processing is not essential for the cellular activity of PT. This distinguishes it from the processing of various other bacterial toxins, which has been shown to be important for their cytotoxicity. S1 processing may be mediated primarily by a metalloprotease, but the cleavage site on S1 is not sequence-dependent and processing appears to depend on the general topology of the protein in that region, indicating that multiple proteases may contribute to this cleavage

Radar sounding evidence for buried glaciers in the southern mid-latitudes of Mars

Lobate features abutting massifs and escarpments in the middle latitudes of Mars have been recognized in images for decades, but their true nature has been controversial, with hypotheses of origin such as ice-lubricated debris flows or glaciers covered by a layer of surface debris. These models imply an ice content ranging from minor and interstitial to massive and relatively pure. Soundings of these deposits in the eastern Hellas region by the Shallow Radar on the Mars Reconnaissance Orbiter reveal radar properties entirely consistent with massive water ice, supporting the debris-covered glacier hypothesis. The results imply that these glaciers formed in a previous climate conducive to glaciation at middle latitudes. Such features may collectively represent the most extensive nonpolar ice yet recognized on Mars

Genetic evidence for the interaction between Bacillus anthracis-encoded phage receptors and their cognate phage-encoded receptor binding proteins

Bacteriophages such as γ and AP50c have been shown to infect strains of Bacillus anthracis with high specificity, and this feature has been exploited in the development of bacterial detection assays. To better understand the emergence of phage resistance, and thus the potential failure of such assays, it is important to identify the host and phage receptors necessary for attachment and entry. Using genetic approaches, the bacterial receptors of AP50c and γ have been identified as sap and GamR, respectively. A second AP50c-like phage, Wip1, also appears to use sap as a receptor. In parallel with this work, the cognate phage-encoded receptor binding proteins (RBPs) have also been identified (Gp14 for γ, P28 for AP50c, and P23 for Wip1); however, the strength of evidence supporting these protein–protein interactions varies, necessitating additional investigation. Here, we present genetic evidence further supporting the interaction between sap and the RBPs of AP50c and Wip1 using fluorescently tagged proteins and a panel of B. anthracis mutants. These results showed that the deletion of the sap gene, as well as the deletion of csaB, whose encoded protein anchors sap to the bacterial S-layer, resulted in the loss of RBP binding. Binding could then be rescued by expressing these genes in trans. We also found that the RBP of the γ-like prophage λBa03 relied on csaB activity for binding, possibly by a different mechanism. RBPλBa03 binding to B. anthracis cells was also unique in that it was not ablated by heat inactivation of vegetative cells, suggesting that its receptor is still functional following incubation at 98°C. These results extend our understanding of the diverse attachment and entry strategies used by B. anthracis phages, enabling future assay development

Whole genome sequencing of phage resistant Bacillus anthracis mutants reveals an essential role for cell surface anchoring protein CsaB in phage AP50c adsorption

BACKGROUND: Spontaneous Bacillus anthracis mutants resistant to infection by phage AP50c (AP50(R)) exhibit a mucoid colony phenotype and secrete an extracellular matrix. METHODS: Here we utilized a Roche/454-based whole genome sequencing approach to identify mutations that are candidates for conferring AP50c phage resistance, followed by genetic deletion and complementation studies to validate the whole genome sequence data and demonstrate that the implicated gene is necessary for AP50c phage infection. RESULTS: Using whole genome sequence data, we mapped the relevant mutations in six AP50(R) strains to csaB. Eleven additional spontaneous mutants, isolated in two different genetic backgrounds, were screened by PCR followed by Sanger sequencing of the csaB gene. In each spontaneous mutant, we found either a non-synonymous substitution, a nonsense mutation, or a frame-shift mutation caused by single nucleotide polymorphisms or a 5 base pair insertion in csaB. All together, 5 and 12 of the 17 spontaneous mutations are predicted to yield altered full length and truncated CsaB proteins respectively. As expected from these results, a targeted deletion or frame-shift mutations introduced into csaB in a different genetic background, in a strain not exposed to AP50c, resulted in a phage resistant phenotype. Also, substitution of a highly conserved histidine residue with an alanine residue (H270A) in CsaB resulted in phage resistance, suggesting that a functional CsaB is necessary for phage sensitivity. Conversely, introduction of the wild type allele of csaB in cis into the csaB deletion mutant by homologous recombination or supplying the wild type CsaB protein in trans from a plasmid restored phage sensitivity. The csaB mutants accumulated cell wall material and appeared to have a defective S-layer, whereas these phenotypes were reverted in the complemented strains. CONCLUSIONS: Taken together, these data suggest an essential role for csaB in AP50c phage infection, most likely in phage adsorption. (The whole genome sequences generated from this study have been submitted to GenBank under SRA project ID: SRA023659.1 and sample IDs: AP50 R1: SRS113675.1, AP50 R2: SRS113676.1, AP50 R3: SRS113728.1, AP50 R4: SRS113733.1, AP50 R6: SRS113734.1, JB220 Parent: SRS150209.1, JB220 Mutant: SRS150211.1)

Improvements to a Markerless Allelic Exchange System for Bacillus anthracis.

A system was previously developed for conducting I-SceI-mediated allelic exchange in Bacillus anthracis. In this system, recombinational loss of a chromosomally-integrated allelic exchange vector is stimulated by creation of a double-stranded break within the vector by the homing endonuclease I-SceI. Although this system is reasonably efficient and represents an improvement in the tools available for allelic exchange in B. anthracis, researchers are nonetheless required to "pick and patch" colonies in order to identify candidate "exchangeants." In the present study, a number of improvements have been made to this system: 1) an improved I-SceI-producing plasmid includes oriT so that both plasmids can now be introduced by conjugation, thus avoiding the need for preparing electro-competent cells of each integration intermediate; 2) antibiotic markers have been changed to allow the use of the system in select agent strains; and 3) both plasmids have been marked with fluorescent proteins, allowing the visualization of plasmid segregation on a plate and obviating the need for "picking and patching." These modifications have made the process easier, faster, and more efficient, allowing for parallel construction of larger numbers of mutant strains. Using this improved system, the genes encoding the tripartite anthrax toxin were deleted singly and in combination from plasmid pXO1 of Sterne strain 34F2. In the course of this study, we determined that DNA transfer to B. anthracis could be accomplished by conjugation directly from a methylation-competent E. coli strain

Utility of fluorescent proteins.

<p>Representative images obtained during the procedure (outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142758#pone.0142758.t001" target="_blank">Table 1</a>) of engineering strain BAP325 (Sterne 34F2 <i>Δlef</i>). Agar plates were imaged with a Fuji LAS-3000 imager. A: pRP1091 in Sterne strain 34F2, imaged under green light (520 nm) on D4 of the procedure, after incubation for 2d at RT. Fluorescent <i>B</i>. <i>anthracis</i> (large, circular colonies) indicates presence of freely replicating pRP1091. B: pRP1091 in 34F2, imaged under green light on D5, after incubation overnight at 37°C. Fluorescence indicates integration of pRP1091 into pXO1. C, D: Images obtained on D10, after transfer of pRP1099 to the integrant via conjugation, under green light (C) and white light (D). Lack of fluorescence (arrowheads) indicates loss of the integrated pRP1091 from pXO1. E, F: Images obtained on D12, after passage in the absence of kanamycin, under blue light (460 nm; E) and white light (F). Lack of fluorescence (arrowheads) indicates loss of pRP1099.</p

Allelic exchange procedure.

<p>Allelic exchange procedure.</p

Improved plasmids for allelic exchange.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142758#sec002" target="_blank">Materials and Methods</a> for descriptions.</p

Description of deletion mutant strains.

<p>Description of deletion mutant strains.</p

Description of plasmid constructs.

<p>Description of plasmid constructs.</p