Bacillus anthracis virulence regulator AtxA: oligomeric state, function and CO(2) -signalling

AtxA, a unique regulatory protein of unknown molecular function, positively controls expression of the major virulence genes of Bacillus anthracis. The 475 amino acid sequence of AtxA reveals DNA binding motifs and regions similar to proteins associated with the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS). We used strains producing native and functional epitope‐tagged AtxA proteins to examine protein–protein interactions in cell lysates and in solutions of purified protein. Co‐affinity purification, non‐denaturing polyacrylamide gel electrophoresis and bis(maleimido)hexane (BMH) cross‐linking experiments revealed AtxA homo‐multimers. Dimers were the most abundant species. BMH cross‐links available cysteines within 13 Å. To localize interaction sites, six AtxA mutants containing distinct Cys→Ser substitutions were tested for multimerization and cross‐linking. All mutants multimerized, but one mutation, C402S, prevented cross‐linking. Thus, BMH uses C402 to make the inter‐molecular bond between AtxA proteins, but C402 is not required for protein–protein interaction. C402 is in a region bearing amino acid similarity to Enzyme IIB proteins of the PTS. The AtxA EIIB motif may function in protein oligomerization. Finally, cultures grown with elevated CO₂/bicarbonate exhibited increased AtxA dimer/monomer ratios and increased AtxA activity, relative to cultures grown without added CO₂/bicarbonate, suggesting that this host‐associated signal enhances AtxA function by shifting the dimer/monomer equilibrium towards the dimeric state.     

The Bicarbonate Transporter Is Essential for Bacillus anthracis Lethality

In the pathogenic bacterium Bacillus anthracis, virulence requires induced expression of the anthrax toxin and capsule genes. Elevated CO₂/bicarbonate levels, an indicator of the host environment, provide a signal ex vivo to increase expression of virulence factors, but the mechanism underlying induction and its relevance in vivo are unknown. We identified a previously uncharacterized ABC transporter (BAS2714-12) similar to bicarbonate transporters in photosynthetic cyanobacteria, which is essential to the bicarbonate induction of virulence gene expression. Deletion of the genes for the transporter abolished induction of toxin gene expression and strongly decreased the rate of bicarbonate uptake ex vivo, demonstrating that the BAS2714-12 locus encodes a bicarbonate ABC transporter. The bicarbonate transporter deletion strain was avirulent in the A/J mouse model of infection. Carbonic anhydrase inhibitors, which prevent the interconversion of CO₂ and bicarbonate, significantly affected toxin expression only in the absence of bicarbonate or the bicarbonate transporter, suggesting that carbonic anhydrase activity is not essential to virulence factor induction and that bicarbonate, and not CO₂, is the signal essential for virulence induction. The identification of this novel bicarbonate transporter essential to virulence of B. anthracis may be of relevance to other pathogens, such as Streptococcus pyogenes, Escherichia coli, Borrelia burgdorferi, and Vibrio cholera that regulate virulence factor expression in response to CO₂/bicarbonate, and suggests it may be a target for antibacterial intervention.     

Petrobactin is the Primary Siderophore Synthesized by <Emphasis Type="BoldItalic">Bacillus anthracis</Emphasis> Str. <Emphasis Type="BoldItalic">Sterne</Emphasis> under Conditions of Iron Starvation

The siderophores of Bacillus anthracis are critical for the pathogen’s proliferation and may be necessary for its virulence. Bacillus anthracis str. Sterne cells were cultured in iron free media and the siderophores produced were isolated and purified using a combination of XAD-2 resin, reverse-phase FPLC, and size exclusion chromatography. A combination of ¹H and ¹³C NMR spectroscopy, UV spectroscopy and ESI-MS/MS fragmentation were used to identify the primary siderophore as petrobactin, a catecholate species containing unusual 3,4-dihydroxybenzoate moieties, previously only identified in extracts of Marinobacter hydrocarbonoclasticus. A secondary siderophore was observed and structural analysis of this species is consistent with that reported for bacillibactin, a siderophore observed in many species of bacilli. This is the first structural characterization of a siderophore from B. anthracis, as well as the first characterization of a 3,4-DHB containing catecholate in a pathogen.     

Ligand and antagonist driven regulation of the Vibrio cholerae quorum-sensing receptor CqsS

Quorum sensing, a bacterial cell–cell communication process, controls biofilm formation and virulence factor production in Vibrio cholerae, a human pathogen that causes the disease cholera. The major V. cholerae autoinducer is (S)‐3‐hydroxytridecan‐4‐one (CAI‐1). A membrane bound two‐component sensor histidine kinase called CqsS detects CAI‐1, and the CqsS → LuxU → LuxO phosphorelay cascade transduces the information encoded in CAI‐1 into the cell. Because the CAI‐1 ligand is known and because the signalling circuit is simple, consisting of only three proteins, this system is ideal for analysing ligand regulation of a sensor histidine kinase. Here we reconstitute the CqsS → LuxU → LuxO phosphorylation cascade in vitro. We find that CAI‐1 inhibits the initial auto‐phosphorylation of CqsS whereas subsequent phosphotransfer steps and CqsS phosphatase activity are not CAI‐1‐controlled. CAI‐1 binding to CqsS causes a conformational change that renders His194 in CqsS inaccessible to the CqsS catalytic domain. CqsS mutants with altered ligand detection specificities are faithfully controlled by their corresponding modified ligands in vitro. Likewise, pairing of agonists and antagonists allows in vitro assessment of their opposing activities. Our data are consistent with a two‐state model for ligand control of histidine kinases.     

Circulating lethal toxin decreases the ability of neutrophils to respond to Bacillus anthracis

Polymorphonuclear leucocytes (PMNs) play a protective role during Bacillus anthracis infection. However, B. anthracis is able to subvert the PMN response effectively as evidenced by the high mortality rates of anthrax. One major virulence factor produced by B. anthracis, lethal toxin (LT), is necessary for dissemination in the BSL2 model of mouse infection. While human and mouse PMNs kill vegetative B. anthracis, short in vitro half‐lives of PMNs have made it difficult to determine how or if LT alters their bactericidal function. Additionally, the role of LT intoxication on PMN's ability to migrate to inflammatory signals remains controversial. LF concentrations in both serum and major organs were determined from mice infected with B. anthracis Sterne strain at defined stages of infection to guide subsequent administration of purified toxin. Bactericidal activity of PMNs assessed using ex vivo cell culture assays showed significant defects in killing B. anthracis. In vivo PMN recruitment to inflammatory stimuli was significantly impaired at 24 h as assessed by real‐time analysis of light‐producing PMNs within the mouse. The observations described above suggest that LT serves dual functions; it both attenuates accumulation of PMNs at sites of inflammation and impairs PMNs bactericidal activity against vegetative B. anthracis.     

Comparative analysis of virulence factors secreted by Bacillus anthracis Sterne at host body temperature

Aims: For the analysis of virulence factors produced and secreted by Bacillus anthracis vegetative cells during mammalian host infection, we evaluated the secretome of B. anthracis Sterne exposed to host‐specific factors specifically to host body temperature. Methods and Results: We employed a comparative proteomics‐based approach to analyse the proteins secreted by B. anthracis Sterne under host‐specific body temperature conditions. A total of 17 proteins encoded on a single chromosome and the pXO1 plasmid were identified by peptide mass fingerprinting. Multiple algorithms were used to predict the secretion mechanisms of the detected proteins in B. anthracis. Conclusions: Several putative virulence factors and known factors responsible for sporulation were differentially regulated, including CodY, pXO1‐130 and BA1952, revealing insights into temperature cues in the B. anthracis secretome. Significance and Impact of the Study: This study identified temperature‐regulated proteins. Further studies aimed at understanding the physical and functional roles of these proteins in infection and control by elevated temperatures will contribute to detection, diagnostics and prophylaxis.     

Cytotoxic Oleanane‐Type Saponins from the Leaves of Albizia anthelmintica Brongn.

Two new oleanane‐type saponins: β‐d ‐xylopyranosyl‐(1 → 4)‐6‐deoxy‐α‐l ‐mannopyranosyl‐(1 → 2)‐1‐O‐{(3β)‐28‐oxo‐3‐[(2‐O‐β‐d ‐xylopyranosyl‐β‐d ‐glucopyranosyl)oxy]olean‐12‐en‐28‐yl}‐β‐d ‐glucopyranose ( 1 ) and 1‐O‐[(3β)‐28‐oxo‐3‐{[β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐arabinopyranosyl‐(1 → 6)‐2‐acetamido‐2‐deoxy‐β‐d ‐glucopyranosyl]oxy}olean‐12‐en‐28‐yl]β‐d ‐glucopyranose ( 2 ), along with two known saponins: (3β)‐3‐[(β‐d ‐Glucopyranosyl‐(1 → 2)‐β‐d ‐glucopyranosyl)oxy]olean‐12‐en‐28‐oic acid ( 3 ) and (3β)‐3‐{[α‐l ‐arabinopyranosyl‐(1 → 6)‐[β‐d ‐glucopyranosyl‐(1 → 2)]‐β‐d ‐glucopyranosyl]oxy}olean‐12‐en‐28‐oic acid ( 4 ) were isolated from the acetone‐insoluble fraction obtained from the 80% aqueous MeOH extract of Albizia anthelmintica Brongn . leaves. Their structures were identified using different NMR experiments including: ¹H‐ and ¹³C‐NMR, HSQC, HMBC and ¹H,¹H‐COSY, together with HR‐ESI‐MS/MS, as well as by acid hydrolysis. The four isolated saponins and the fractions of the extract exhibited cytotoxic activity against HepG‐2 and HCT‐116 cell lines. Compound 2 showed the most potent cytotoxic activity among the other tested compounds against the HepG2 cell line with an IC₅₀ value of 3.60μm . Whereas, compound 1 showed the most potent cytotoxic effect with an IC₅₀ value of 4.75μm on HCT‐116 cells.     

Regulation of the Escherichia coli antiterminator protein BglG by phosphorylation at multiple sites and evidence for transfer of phosphoryl groups between monomers

Activity of antiterminator protein BglG regulating the beta-glucoside operon in Escherichia coli is controlled by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in a dual manner. It requires HPr phosphorylation to be active, whereas phosphorylation by the beta-glucoside-specific transport protein EIIBgl inhibits its activity. BglG and its relatives carry two PTS regulation domains (PRD1 and PRD2), each containing two conserved histidines. For BglG, histidine 208 in PRD2 was reported to be the negative phosphorylation site. In contrast, other antiterminators of this family are negatively regulated by phosphorylation of the first histidine in PRD1, and presumably activated by phosphorylation of the histidines in PRD2. In this work, a screen for mutant BglG proteins that escape repression by EIIBgl yielded exchanges of nine residues within PRD1, including conserved histidines His-101 and His-160, and C-terminally truncated proteins. Genetic and phosphorylation analyses indicate that His-101 in PRD1 is phosphorylated by EIIBgl and that His-160 contributes to negative regulation. His-208 in PRD2 is essential for BglG activity, suggesting that it is phosphorylated by HPr. Surprisingly, phosphorylation by HPr is not fully abolished by exchanges of His-208. However, phosphorylation by HPr is inhibited by exchanges in PRD1 and the phosphorylation of these mutants is restored in the presence of wild-type BglG. These results suggest that the activating phosphoryl group is transiently donated from HPr to PRD1 and subsequently transferred to His-208 of a second BglG monomer. The active His-208-phosphorylated BglG dimer can subsequently be inhibited in its activity by EIIBgl-catalyzed phosphorylation at His-101.     

A neurotoxic phospholipase a(2) impairs yeast amphiphysin activity and reduces endocytosis

Background

Presynaptically neurotoxic phospholipases A₂ inhibit synaptic vesicle recycling through endocytosis.

Principal Findings

Here we provide insight into the action of a presynaptically neurotoxic phospholipase A₂ ammodytoxin A (AtxA) on clathrin-dependent endocytosis in budding yeast. AtxA caused changes in the dynamics of vesicle formation and scission from the plasma membrane in a phospholipase activity dependent manner. Our data, based on synthetic dosage lethality screen and the analysis of the dynamics of sites of endocytosis, indicate that AtxA impairs the activity of amphiphysin.

Conclusions

We identified amphiphysin and endocytosis as the target of AtxA intracellular activity. We propose that AtxA reduces endocytosis following a mechanism of action which includes both a specific protein–protein interaction and enzymatic activity, and which is applicable to yeast and mammalian cells. Knowing how neurotoxic phospholipases A₂ work can open new ways to regulate endocytosis.     

Toxin-Independent Virulence of Bacillus anthracis in Rabbits

The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play a major role in pathogenicity. In the guinea pig (GP) model we have previously shown that deletion of all three toxin components results in a relatively moderate attenuation in virulence, indicating that B. anthracis possesses an additional toxin-independent virulence mechanism. To characterize this toxin-independent mechanism in anthrax disease, we developed a new rabbit model by intravenous injection (IV) of B. anthracis encapsulated vegetative cells, artificially creating bacteremia. Using this model we were able to demonstrate that also in rabbits, B. anthracis mutants lacking the toxins are capable of killing the host within 24 hours. This virulent trait depends on the activity of AtxA in the presence of pXO2, as, in the absence of the toxin genes, deletion of either component abolishes virulence. Furthermore, this IV virulence depends mainly on AtxA rather than the whole pXO1. A similar pattern was shown in the GP model using subcutaneous (SC) administration of spores of the mutant strains, demonstrating the generality of the phenomenon. The virulent strains showed higher bacteremia levels and more efficient tissue dissemination; however our interpretation is that tissue dissemination per se is not the main determinant of virulence whose exact nature requires further elucidation.     

Triterpene Glucosides from the Leaves of Aralia elata and Their Cytotoxic Activities

Three new triterpene glucosides, named congmuyenosides C–E ( 1 – 3 , resp.), along with four known ones, were isolated from an EtOH extract of Aralia elata (Miq .) Seem . leaves. The structures of the new compounds were identified as 3‐O‐{β‐D ‐glucopyranosyl‐(1→3)‐β‐D ‐glucopyranosyl‐(1→3)‐[β‐D ‐glucopyranosyl‐(1→2)]‐β‐D ‐glucopyranosyl}caulophyllogenin ( 1 ), 3‐O‐{β‐D ‐glucopyranosyl‐(1→3)‐β‐D ‐glucopyranosyl‐(1→3)‐[β‐D ‐glucopyranosyl‐(1→2)]‐β‐D ‐glucopyranosyl}hederagenin 28‐O‐β‐D ‐glucopyranosyl ester ( 2 ), 3‐O‐{β‐D ‐glucopyranosyl‐(1→3)‐β‐D ‐glucopyranosyl‐(1→3)‐[β‐D ‐glucopyranosyl‐(1→2)]‐β‐D ‐glucopyranosyl}echinocystic acid 28‐O‐β‐D ‐glucopyranosyl ester ( 3 ) on the basis of spectral analyses, including MS, ¹H‐NMR, ¹³C‐NMR, DEPT, HSQC, HMBC, NOESY, and HSQC‐TOCSY experiments. All isolates obtained were evaluated for their cytotoxic activities against three human tumor cell lines (HepG2, SKOV3, and A549). Compound 3 showed significant cytotoxicity against A549 cell line (IC₅₀ 9.9±1.5 μM ).