Inhibition of Bacillus anthracis metallo-β-lactamase by compounds with hydroxamic acid functionality

Abstract

Metallo-β-lactamases (MBLs) that catalyze hydrolysis of β-lactam antibiotics are an emerging threat due to their rapid spread. A strain of the bacterium Bacillus anthracis has its ability to produce and secrete a MBL, referred to Bla2. To address this challenge, novel hydroxamic acid-containing compounds such as 3-(heptyloxy)-N-hydroxybenzamide (compound 4) and N-hydroxy-3-((6-(hydroxyamino)-6-oxohexyl)oxy)benzamide (compound 7) were synthesized. Kinetic analysis of microbial inhibition indicated that the both sides of hydroxamic acids containing compound 7 revealed a reversible, competitive inhibition with a K_i value of 0.18?±?0.06?μM. The result has reflected that the both sides of dihydroxamic acids in a molecule play a crucial role in the binding affinity rather than monohydroxamic containing compound 4 which was unable to inhibit Bla2. In addition, in silico analysis suggested that compound 7 was coordinated with a zinc ion in the active site of enzyme. These observations suggest that the dihydroxamic acid-containing compound may be a promising drug candidate, and a further implication for designing new inhibitors of Bla2.     

炭疽芽胞杆菌(Bacillus anthracis)检测质粒的构建及其应用

根据炭疽芽胞杆菌(Bacillus anthracis)毒性质粒pX01和pX02上的2个毒力相关基因cya和capA的序列特点,以pIJ2925为出发载体,采用一步重叠延伸PCR技术(One-step Overlap Extension PCR,简称OOE-PCR)构建了包含cya基因和capA基因保守区DNA片段的炭疽检测质粒pBIB2006。采用复合PCR对模拟炭疽危险品进行分析,结果表明pBIB2006可以为炭疽芽胞杆菌的检测提供准确、安全和方便的阳性参照品,从而为检测炭疽芽胞杆菌和炭疽芽胞杆菌灭活疫苗提供了便利。     

Ecological Niche Modeling of Bacillus anthracis on Three Continents: Evidence for Genetic-Ecological Divergence?

We modeled the ecological niche of a globally successful Bacillus anthracis sublineage in the United States, Italy and Kazakhstan to better understand the geographic distribution of anthrax and potential associations between regional populations and ecology. Country-specific ecological-niche models were developed and reciprocally transferred to the other countries to determine if pathogen presence could be accurately predicted on novel landscapes. Native models accurately predicted endemic areas within each country, but transferred models failed to predict known occurrences in the outside countries. While the effects of variable selection and limitations of the genetic data should be considered, results suggest differing ecological associations for the B. anthracis populations within each country and may reflect niche specialization within the sublineage. Our findings provide guidance for developing accurate ecological niche models for this pathogen; models should be developed regionally, on the native landscape, and with consideration to population genetics. Further genomic analysis will improve our understanding of the genetic-ecological dynamics of B. anthracis across these countries and may lead to more refined predictive models for surveillance and proactive vaccination programs. Further studies should evaluate the impact of variable selection of native and transferred models.     

Poly-γ-d-Glutamic Acid Capsule Interferes with Lytic Infection of Bacillus anthracis by B. anthracis-Specific Bacteriophages

The poly-γ-d-glutamic acid capsule of Bacillus anthracis is a barrier to infection by B. anthracis-specific bacteriophages. Capsule expression was found to completely inhibit lytic infection by γ phage, an observation supported by the demonstration that this phage does not elaborate a hydrolase that would facilitate penetration through the protective capsule outer layer.     

Evidence that YycJ is a novel 5′–3′ double-stranded DNA exonuclease acting in Bacillus anthracis mismatch repair

The most important system for correcting replication errors that survive the built in editing system of DNA polymerase is the mismatch repair (MMR) system. We have identified a novel mutator strain yycJ in Bacillus anthracis. Mutations in the yycJ gene result in a spontaneous mutator phenotype with a mutational frequency and specificity comparable to that of MMR-deficient strains such as those with mutations in mutL or mutS. YycJ was annotated as a metallo-β-lactamase (MβL) super family member with unknown activity. In this study we carried out a biochemical characterization of YycJ and demonstrated that a recombinant YycJ protein possesses a 5′–3′ exonuclease activity at the 5′ termini and at nicks of double-stranded DNA. This activity requires a divalent metal cofactor Mn²⁺ and is stimulated by 5′-phosphate ends of duplex DNA. The mutagenesis of conserved amino acid residues revealed that in addition to the five MβL family conserved motifs, YycJ appears to have its specific motifs that can be used to distinguish YycJ from other closely related MβL family members. A phylogenetic survey showed that putative YycJ homologs are present in several bacterial phyla as well as in members of the Methanomicrobiales and Thermoplasmales from Archaea. We propose that YycJ represents a new group of MβL fold exonucleases, which is likely to act in the recognition of MMR entry point and subsequent removal of the mismatched base in certain MutH-less bacterial species.     

Rapid Identification of Bacillus anthracis Spores in Suspicious Powder Samples by Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS)

Rapid and reliable identification of Bacillus anthracis spores in suspicious powders is important to mitigate the safety risks and economic burdens associated with such incidents. The aim of this study was to develop and validate a rapid and reliable laboratory-based matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis method for identifying B. anthracis spores in suspicious powder samples. A reference library containing 22 different Bacillus sp. strains or hoax materials was constructed and coupled with a novel classification algorithm and standardized processing protocol for various powder samples. The method''s limit of B. anthracis detection was determined to be 2.5 × 10⁶ spores, equivalent to a 55-μg sample size of the crudest B. anthracis-containing powder discovered during the 2001 Amerithrax incidents. The end-to-end analysis method was able to successfully discriminate among samples containing B. anthracis spores, closely related Bacillus sp. spores, and commonly encountered hoax materials. No false-positive or -negative classifications of B. anthracis spores were observed, even when the analysis method was challenged with a wide range of other bacterial agents. The robustness of the method was demonstrated by analyzing samples (i) at an external facility using a different MALDI-TOF MS instrument, (ii) using an untrained operator, and (iii) using mixtures of Bacillus sp. spores and hoax materials. Taken together, the observed performance of the analysis method developed demonstrates its potential applicability as a rapid, specific, sensitive, robust, and cost-effective laboratory-based analysis tool for resolving incidents involving suspicious powders in less than 30 min.     

Entry of Bacillus anthracis spores into epithelial cells is mediated by the spore surface protein BclA, integrin α2β1 and complement component C1q

Inhalational anthrax is initiated by pulmonary exposure to Bacillus anthracis spores. Spore entry into lung epithelial cells is observed both in vitro and in vivo and evidence suggests it is important for bacterial dissemination and virulence. However the specific host receptor and spore factor that mediate the entry process were unknown. Here, we report that integrin α2β1 is a major receptor for spore entry. This is supported by results from blocking antibodies, siRNA knock-down, colocalization, and comparison of spore entry into cells that do or do not express α2. BclA, a major spore surface protein, is found to be essential for entry and α2β1-mediated entry is dependent on BclA. However, BclA does not appear to bind directly to α2. Furthermore, spore entry into α2-expressing cells is dramatically reduced in the absence of serum, suggesting that additional factors are involved. Finally, complement component C1q, also an α2β1 ligand, appears to act as a bridging molecule or a cofactor for BclA/α2β1-mediated spore entry and BclA binds to C1q in a dose-dependent and saturable manner. These findings suggest a novel mechanism for pathogen entry into host cells as well as a new function for C1q-integrin interactions. The implications of these findings are discussed.