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1.
Nat Commun ; 11(1): 840, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-32047164

RESUMO

Following assembly, the anthrax protective antigen (PA) forms an oligomeric translocon that unfolds and translocates either its lethal factor (LF) or edema factor (EF) into the host cell. Here, we report the cryo-EM structures of heptameric PA channels with partially unfolded LF and EF at 4.6 and 3.1-Å resolution, respectively. The first α helix and ß strand of LF and EF unfold and dock into a deep amphipathic cleft, called the α clamp, which resides at the interface of two PA monomers. The α-clamp-helix interactions exhibit structural plasticity when comparing the structures of lethal and edema toxins. EF undergoes a largescale conformational rearrangement when forming the complex with the channel. A critical loop in the PA binding interface is displaced for about 4 Å, leading to the weakening of the binding interface prior to translocation. These structures provide key insights into the molecular mechanisms of translocation-coupled protein unfolding and translocation.


Assuntos
Antígenos de Bactérias/química , Toxinas Bacterianas/química , Desdobramento de Proteína , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Bacillus anthracis/genética , Bacillus anthracis/metabolismo , Toxinas Bacterianas/genética , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas
2.
BMC Infect Dis ; 20(1): 140, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-32059712

RESUMO

BACKGROUND: Anthrax is an endemic disease that persists in the rural regions of China. The global genetic population structure of B.anthracis has also been defined by the canonical single-nucleotide polymorphisms (canSNP) and multiple-locus variable-number tandem repeat analysis (MLVA). Five canSNP lineages were found in China, and the A.Br.Ames lineage has been the second predominant group in recent years in China. The objective of this study was to reveal genetic diversity of the Ames lineage strains by MLVA. METHODS: Two molecular typing methods, canSNP and MLVA with 15markers were used to study the genetic relationship among the Ames lineage strains. The outbreak information associated with these strains was also collected and investigated. RESULTS: From 2007 to 2018, a total of 21 human anthrax infection outbreaks (68 patients) associated with B. anthracis Ames lineage strains were reported in China. Ames lineage strain-associated human anthrax is mainly distributed in the northern part of China, including the provinces of Inner Mongolia, Liaoning, Gansu, and Xinjiang. In the study, a total of 30 Ames lineage strains were included and 10 MLVA15 genotypes were identified. These strains were mainly found in northeast China, Inner Mongolia and Liaoning. In recent years, the Ames lineage strains were isolated in the two provinces every year. The 18 Ames lineage strains isolated from Inner Mongolia were divided into eight MLVA15 genotypes. From 2010 to 2015, there were continuous reports of outbreaks in Keyouzhongqi County, Inner Mongolia, and the strains that were isolated annually in succession belonged to the MLVA15-30 genotype. CONCLUSIONS: The Ames lineage strains are widely distributed in northern China. Their genetic diversity can be illustrated by the results of the MLVA. The genetic characteristics of the Ames lineage strains from outbreaks in different provinces varied. In some areas, human anthrax outbreaks occurred annually in succession, and these related strains grouped together. These observations indicate that the local environment was persistently contaminated with B. anthracis spores, vaccination of livestock should become the fundamental control measure in the areas.


Assuntos
Antraz/microbiologia , Bacillus anthracis/genética , Variação Genética , Animais , Antraz/epidemiologia , Bacillus anthracis/isolamento & purificação , China/epidemiologia , Surtos de Doenças , Genética Populacional , Genótipo , Humanos , Gado/microbiologia , Repetições Minissatélites , Tipagem Molecular , Polimorfismo de Nucleotídeo Único
3.
PLoS One ; 15(2): e0228116, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084143

RESUMO

To understand the epidemiological and genetic background of anthrax cases occurring in Vietnam from 2011 to 2015, we surveilled and genetically analyzed Bacillus anthracis isolated in the north of the country. Epidemiological surveillance showed that most human cutaneous anthrax cases occurred in association with animal dissection. Whole-genome sequences were obtained from six B. anthracis strains from human patients with cutaneous anthrax in the endemic area. Comparative genomic analysis showed that the genetic homogeneity among Vietnamese B. anthracis strains was very high. All Vietnamese B. anthracis strains belonged to the canSNP lineage of A.Br.011/009, which mostly consists of strains of the trans-Eurasian (TEA) group, including the most closely related strain, Carbosap. To clarify the genetic diversity of Vietnamese strains and strains belonging to A.Br.011/009 and A.Br.008/011 canSNP lineages, we applied a reference genome-based single-nucleotide polymorphism (SNP) and gene-by-gene genomic analysis (whole-genome MLST) strategy. The phylogeny from core genome SNPs revealed that the Vietnamese strains were positioned close to each other; moreover, several SNPs specific to Vietnamese B. anthracis were identified. Whole-genome MLST analysis revealed the differences in the number of SNPs between Vietnamese strains, which could enable discrimination at the strain level.


Assuntos
Antraz/epidemiologia , Bacillus anthracis/genética , Genômica , Dermatopatias Bacterianas/epidemiologia , Antraz/microbiologia , Bacillus anthracis/isolamento & purificação , Bacillus anthracis/fisiologia , Genoma Bacteriano/genética , Humanos , Tipagem de Sequências Multilocus , Filogenia , Polimorfismo de Nucleotídeo Único , Dermatopatias Bacterianas/microbiologia , Vietnã/epidemiologia
4.
PLoS One ; 15(1): e0227875, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31931511

RESUMO

In Italy anthrax is an endemic disease, with a few outbreaks occurring almost every year. We surveyed 234 B. anthracis strains from animals (n = 196), humans (n = 3) and the environment (n = 35) isolated during Italian outbreaks in the years 1972-2018. Despite the considerable genetic homogeneity of B. anthracis, the strains were effectively differentiated using canonical single nucleotide polymorphisms (CanSNPs) assay and multiple-locus variable-number tandem repeat analysis (MLVA). The phylogenetic identity was determined through the characterization of 14 CanSNPs. In addition, a subsequent 31-loci MLVA assay was also used to further discriminate B. anthracis genotypes into subgroups. The analysis of 14 CanSNPs allowed for the identification of four main lineages: A.Br.011/009, A.Br.008/011 (respectively belonging to A.Br.008/009 sublineage, also known Trans-Eurasian or TEA group), A.Br.005/006 and B.Br.CNEVA. A.Br.011/009, the most common subgroup of lineage A, is the major genotype of B. anthracis in Italy. The MLVA analysis revealed the presence of 55 different genotypes in Italy. Most of the genotypes are genetically very similar, supporting the hypothesis that all strains evolved from a local common ancestral strain, except for two genotypes representing the branch A.Br.005/006 and B.Br.CNEVA. The genotyping analysis applied in this study remains a very valuable tool for studying the diversity, evolution, and molecular epidemiology of B. anthracis.


Assuntos
Antraz/genética , Bacillus anthracis/genética , Epidemiologia Molecular , Filogenia , Animais , Antraz/epidemiologia , Antraz/microbiologia , Bacillus anthracis/classificação , Bacillus anthracis/patogenicidade , Genoma Bacteriano/genética , Genótipo , Humanos , Itália/epidemiologia , Repetições Minissatélites/genética , Polimorfismo de Nucleotídeo Único/genética
5.
Ann Agric Environ Med ; 26(3): 392-395, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31559791

RESUMO

Existing research for using the protective antigen (PA) of Bacillus anthracis as a vaccine component shows that protection against anthrax may be obtained using fragments of this protein. The aim of the research is to check whether the selected protein fragment of the protective antigen (domain 4) encoded by an appropriate nucleotide sequence of gene pag of B. anthracis, was expressed in the bacterial system of E. coli. In order to examine the selected sequence of the pag gene, a PCR reaction and a highly effective TOPO cloning strategy were used, followed by purification of the recombinant proteins and their detection by a western-blot method. In the planning of the PA4 antigen expression a higher level of effectiveness in production of small protein - domain 4 - was anticipated. As a result, the 139 amino acids protein fragment of B. anthracis PA (domain 4) was isolated. The research may have found the basis for in vivo research aimed at finding potential anthrax vaccine components.


Assuntos
Vacinas contra Antraz/imunologia , Antraz/microbiologia , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Toxinas Bacterianas/imunologia , Animais , Antraz/imunologia , Antraz/prevenção & controle , Vacinas contra Antraz/administração & dosagem , Vacinas contra Antraz/genética , Vacinas contra Antraz/isolamento & purificação , Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Bacillus anthracis/química , Bacillus anthracis/genética , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Western Blotting , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Domínios Proteicos
6.
BMC Genomics ; 20(1): 692, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477029

RESUMO

BACKGROUND: Anthrax is a zoonotic disease caused by the gram-positive bacterium Bacillus anthracis. The most anthrax-endemic regions of Russia are Siberia and North Caucasus. Previously, genotyping of Russian B.anthracis isolates was carried out using canSNP and MLVA data; these methods yield lower resolution results compared to whole genome SNP analysis (wgSNP). In this research, we have used wgSNP method for genotyping of 10 B.anthracis isolates, obtained during 1961-2016 in Russia on territory of Western Siberia. RESULTS: We have analyzed 185 B.anthracis genomes available in GenBank database and genomes of 10 isolates obtained in this study to determine the place of Russian isolates in the global phylogeny of B.anthracis. For the studied genomes we have detected 7203 SNPs, which were used for building a phylogenetic reconstruction with Maximum Likelihood Method. Results of the phylogenetic analysis indicate that Russian strains belong to three different genetic groups. Three strains belong to genetic group "Ames", two strains - to "STI" group. Five strains belong to the main genetic line B, and four of them form a subcluster, described for the first time, which we have named "Siberia". CONCLUSIONS: In this study, the data on genetic diversity of B.anthracis strains on the territory of Western Siberia is presented for the first time. As a result of complex phylogenetic analysis, the place of these isolates was determined in the global phylogenetic structure of the B.anthracis population. We describe a new cluster in the main genetic line B for the first time.


Assuntos
Bacillus anthracis/genética , Filogenia , Bacillus anthracis/classificação , Família Multigênica , Polimorfismo de Nucleotídeo Único , Sibéria , Sequenciamento Completo do Genoma
7.
PLoS Negl Trop Dis ; 13(8): e0007644, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31430284

RESUMO

Bacillus anthracis and Yersinia pestis are zoonotic bacteria capable of causing severe and sometimes fatal infections in animals and humans. Although considered as diseases of antiquity in industrialized countries due to animal and public health improvements, they remain endemic in vast regions of the world disproportionally affecting the poor. These pathogens also remain a serious threat if deployed in biological warfare. A single vaccine capable of stimulating rapid protection against both pathogens would be an extremely advantageous public health tool. We produced multiple-antigen fusion proteins (MaF1 and MaF2) containing protective regions from B. anthracis protective antigen (PA) and lethal factor (LF), and from Y. pestis V antigen (LcrV) and fraction 1 (F1) capsule. The MaF2 sequence was also expressed from a plasmid construct (pDNA-MaF2). Immunogenicity and protective efficacy were investigated in mice following homologous and heterologous prime-boost immunization. Antibody responses were determined by ELISA and anthrax toxin neutralization assay. Vaccine efficacy was determined against lethal challenge with either anthrax toxin or Y. pestis. Both constructs elicited LcrV and LF-specific serum IgG, and MaF2 elicited toxin-neutralizing antibodies. Immunizations with MaF2 conferred 100% and 88% protection against Y. pestis and anthrax toxin, respectively. In contrast, pDNA-MaF2 conferred only 63% protection against Y. pestis and no protection against anthrax toxin challenge. pDNA-MaF2-prime MaF2-boost induced 75% protection against Y. pestis and 25% protection against anthrax toxin. Protection was increased by the molecular adjuvant CARDif. In conclusion, MaF2 is a promising multi-antigen vaccine candidate against anthrax and plague that warrants further investigation.


Assuntos
Antraz/prevenção & controle , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Vacinas Bacterianas/imunologia , Peste/prevenção & controle , Proteínas Recombinantes de Fusão/imunologia , Yersinia pestis/imunologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Bacillus anthracis/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Vacinas Bacterianas/isolamento & purificação , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos BALB C , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/imunologia , Proteínas Recombinantes de Fusão/genética , Análise de Sobrevida , Resultado do Tratamento , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Yersinia pestis/genética
8.
Indian J Med Microbiol ; 37(1): 116-119, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31424022

RESUMO

Aims and Objectives: Molecular confirmation of the circulating Bacillus anthracis during outbreak of anthrax in different villages of Simdega district, Jharkhand, India. Materials and Methods: Blood samples with swabs from skin lesions (eschar) were collected from the suspected cases of Anthrax from October 2014 to June 2016 from Simdega district, Jharkhand. All the swabs were inoculated on polymyxin lysozyme EDTA thallous acetate media, nutrient agar media as well as 5% sheep blood agar media. Gamma-phage lysis was done. DNA extraction was done using a QIAamp DNA Mini Kit (QIAGEN, Valencia, CA, USA) and subjected to polymerase chain reaction (PCR) using anthrax-specific primers. Results: On Gram and acid fast staining, purple rods and pink-coloured anthrax spores were detected. Capsular and M'Fadyean staining was done. Gamma-phage lysed B. anthracis culture. Of 39 suspected cases, 8 were culture and PCR positive and showed gamma-phage lysis. 3 deaths were reported. Discussion and Conclusion: The conventional and real-time PCR methods are suitable for both the clinical and the epidemiological practice.


Assuntos
Antraz/diagnóstico , Antraz/epidemiologia , Bacillus anthracis/genética , Dermatopatias Bacterianas/diagnóstico , Dermatopatias Bacterianas/epidemiologia , Esporos Bacterianos/isolamento & purificação , Adulto , Antraz/tratamento farmacológico , Antibacterianos/uso terapêutico , Bacillus anthracis/isolamento & purificação , Ciprofloxacino/uso terapêutico , Surtos de Doenças , Humanos , Índia/epidemiologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Dermatopatias Bacterianas/tratamento farmacológico , Adulto Jovem
9.
Biologicals ; 61: 38-43, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31416791

RESUMO

Tremendous efforts are being made to develop an anthrax vaccine with long term protection. The main component of traditional anthrax vaccine is protective antigen (PA) with the trace amount of other proteins and bacterial components. In this study, we developed a recombinant PA-LF chimera antigen of Bacillus anthracis by fusing the PA domain 2-4 with lethal factor (LF) domain 1 and evaluated its protective potential against B. anthracis in mouse model. The anti-PA-LF chimera serum reacted with both PA and LF antigen, individually. The chimera elicited a strong antibody titer in mice with predominance of IgG1 isotype followed by IgG2b, IgG2a and IgG3. Cytokines were assessed in splenocytes of immunized mice and a significant up-regulation in the expression of IL-4, IL-10, IFN-γ and TNF-α was observed. The PA-LF chimera immunized mice exhibited 80% survival after challenge with virulent spores of B. anthracis. Pathological studies showed normal architecture in vital organs (spleen, lung, liver and kidney) of recovered immunized mice on 20 DPI after spore challenge. These findings suggested that PA-LF chimera of B. anthracis elicited good humoral as well as cell mediated immune response in mice, and thus, can be a potent vaccine candidate against anthrax.


Assuntos
Vacinas contra Antraz/imunologia , Antraz/prevenção & controle , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Toxinas Bacterianas/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Antraz/imunologia , Antraz/patologia , Vacinas contra Antraz/genética , Antígenos de Bactérias/genética , Bacillus anthracis/genética , Toxinas Bacterianas/genética , Gerenciamento Clínico , Avaliação de Medicamentos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética
10.
BMC Vet Res ; 15(1): 265, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31357988

RESUMO

BACKGROUND: Anthrax caused by Bacillus anthracis is a zoonotic disease mainly affecting herbivores. The last Swiss outbreak was over 20 years ago. We describe a recent anthrax outbreak involving two cows from the same herd. One cow was designated as a peracute clinical case with sudden death and typical lung lesions, while the other cow presented with protracted fever and abortion. CASE PRESENTATION: On April 29th 2017, a 3.5-year-old Montbéliard dairy cow was found dead while out at pasture with haemorrhage from the nose. The veterinarian suspected pneumonia and performed a necropsy on site. Subsequently, a lung and liver sample were sent to the laboratory. Unexpectedly, Bacillus anthracis was isolated, a pathogen not found in Switzerland for decades. Several days later, a second cow from the same farm showed signs of abortion after protracted fever. Since these symptoms are not typical for anthrax, and the bacteria could not be demonstrated in blood samples from this animal, a necropsy was performed under appropriate biosafety measures. Subsequently, Bacillus anthracis could be isolated from the placenta and the sublumbal lymph nodes but not from the blood, liver, spleen and kidney. The outbreak strain (17OD930) was shown to belong to the lineage B.Br.CNEVA, the same as Swiss strains from previous outbreaks in the region. We speculate that the disease came from a temporarily opened cave system that is connected to an old carcass burial site and was flushed by heavy rainfall preceding the outbreak. CONCLUSION: Even in countries like Switzerland, where anthrax is very rare, new cases can occur after unusual weather conditions or ground disturbance. It is important for public officials to be aware of this risk to avoid possible spread.


Assuntos
Antraz/veterinária , Doenças dos Bovinos/patologia , Aborto Animal/etiologia , Animais , Antraz/complicações , Antraz/microbiologia , Antraz/patologia , Bacillus anthracis/classificação , Bacillus anthracis/genética , Bacillus anthracis/isolamento & purificação , Bovinos , Doenças dos Bovinos/microbiologia , Cavernas/microbiologia , Feminino , Gravidez , Fatores de Risco , Suíça , Tempo (Meteorologia)
11.
Mol Microbiol ; 112(2): 515-531, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31063630

RESUMO

A challenge common to all bacterial pathogens is to acquire nutrients from hostile host environments. Iron is an important cofactor required for essential cellular processes such as DNA repair, energy production and redox balance. Within a mammalian host, most iron is sequestered within heme, which in turn is predominantly bound by hemoglobin. While little is understood about the mechanisms by which bacterial hemophores attain heme from host-hemoglobin, even less is known about intracellular heme processing. Bacillus anthracis, the causative agent of anthrax, displays a remarkable ability to grow in mammalian hosts. Hypothesizing this pathogen harbors robust ways to catabolize heme, we characterize two new intracellular heme-binding proteins that are distinct from the previously described IsdG heme monooxygenase. The first of these, HmoA, binds and degrades heme, is necessary for heme detoxification and facilitates growth on heme iron sources. The second protein, HmoB, binds and degrades heme too, but is not necessary for heme utilization or virulence. The loss of both HmoA and IsdG renders B. anthracis incapable of causing anthrax disease. The additional loss of HmoB in this background increases clearance of bacilli in lungs, which is consistent with this protein being important for survival in alveolar macrophages.


Assuntos
Antraz/microbiologia , Bacillus anthracis/metabolismo , Heme/metabolismo , Antraz/metabolismo , Bacillus anthracis/enzimologia , Bacillus anthracis/genética , Bacillus anthracis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Ligação Proteica
12.
PLoS One ; 14(5): e0209140, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31116737

RESUMO

This article describes Bacillus anthracis strains isolated during an outbreak of anthrax on the Yamal Peninsula in the summer of 2016 and independently in Yakutia in 2015. A common feature of these strains is their conservation in permafrost, from which they were extracted either due to the thawing of permafrost (Yamal strains) or as the result of paleontological excavations (Yakut strains). All strains isolated on the Yamal share an identical genotype belonging to lineage B.Br.001/002, pointing to a common source of infection in a territory over 250 km in length. In contrast, during the excavations in Yakutia, three genetically different strains were recovered from a single pit. One strain belongs to B.Br.001/002, and whole genome sequence analysis showed that it is most closely related to the Yamal strains in spite of the remoteness of Yamal from Yakutia. The two other strains contribute to two different branches of A.Br.008/011, one of the remarkable polytomies described so far in the B. anthracis species. The geographic distribution of the strains belonging to A.Br.008/011 is suggesting that the polytomy emerged in the thirteenth century, in combination with the constitution of a unified Mongol empire extending from China to Eastern Europe. We propose an evolutionary model for B. anthracis recent evolution in which the B lineage spread throughout Eurasia and was subsequently replaced by the A lineage except in some geographically isolated areas.


Assuntos
Antraz/epidemiologia , Antraz/microbiologia , Bacillus anthracis/classificação , Bacillus anthracis/genética , Pergelissolo/microbiologia , Microbiologia do Solo , Animais , Antraz/transmissão , Bacillus anthracis/isolamento & purificação , Surtos de Doenças , Genoma Bacteriano , Genômica/métodos , Cobaias , Humanos , Camundongos , Filogenia , Polimorfismo de Nucleotídeo Único , Federação Russa/epidemiologia
13.
Infect Genet Evol ; 71: 128-139, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30928604

RESUMO

Anthrax, caused by Bacillus anthracis, is a severe zoonosis with a great impact on both human and animal health. In the present study, we identified the phylogenetic relationships among 16 Japanese strains of B. anthracis, including eight bovine strains, two equine strains, five swine strains, and one former vaccine strain, using in silico canonical single nucleotide polymorphism (canSNP) and core genome SNP analyses. The results of our in silico canSNP analysis suggest that these 16 Japanese strains could be divided into four lineages: i) one equine strain in A.Br.Ames, ii) one equine and six bovine strains in A.Br.001/002, iii) five swine and one bovine strain in A.Br.Aust94, and iv) one bovine and one vaccine strain in A.Br.008/011. A comparison with non-Japanese B. anthracis strains revealed a total of 3787 SNPs identified from the whole genome sequences of the Japanese strains; these SNP data were subjected to a phylogenetic analysis using the maximum parsimony (MP) method. Our core genome SNP analysis was also able to detect differences of a few chromosomal SNPs across clonal strains from the same cases that had different storage and passage histories. Additionally, our whole genome SNP analysis clearly indicated that the Japanese swine anthrax cases of 1982 were caused by at least three independent strains; however, their phylogeny revealed no clear relationship with swine strains from other countries. The bovine strain belonging to the A.Br.008/011 lineage differed from a former Japanese vaccine strain by only 12 SNPs. Together with the phylogenic results and epidemiological circumstances, the diversity of strains reveals that the B. anthracis available in Japan probably resulted from multiple relatively recent import events, rather than reflecting the persistence of a more ancient ecologically established group.


Assuntos
Antraz/veterinária , Bacillus anthracis/classificação , Bacillus anthracis/genética , Animais , Animais Domésticos/microbiologia , Bovinos , Biologia Computacional , Genoma Bacteriano , Genômica , Cavalos , Japão/epidemiologia , Epidemiologia Molecular , Filogenia , Filogeografia , Polimorfismo de Nucleotídeo Único , Suínos , Sequenciamento Completo do Genoma
14.
Appl Environ Microbiol ; 85(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30658980

RESUMO

Acylation of epsilon amino groups of lysyl side chains is a widespread modification of proteins and small molecules in cells of all three domains of life. Recently, we showed that Bacillus subtilis and Bacillus anthracis encode the GCN5-related N-acetyltransferase (GNAT) SatA that can acetylate and inactivate streptothricin, which is a broad-spectrum antibiotic produced by actinomycetes in the soil. To determine functionally relevant residues of B. subtilis SatA (BsSatA), a mutational screen was performed, highlighting the importance of a conserved area near the C terminus. Upon inspection of the crystal structure of the B. anthracis Ames SatA (BaSatA; PDB entry 3PP9), this area appears to form a pocket with multiple conserved aromatic residues; we hypothesized this region contains the streptothricin-binding site. Chemical and site-directed mutagenesis was used to introduce missense mutations into satA, and the functionality of the variants was assessed using a heterologous host (Salmonella enterica). Results of isothermal titration calorimetry experiments showed that residue Y164 of BaSatA was important for binding streptothricin. Results of size exclusion chromatography analyses showed that residue D160 was important for dimerization. Together, these data advance our understanding of how SatA interacts with streptothricin.IMPORTANCE This work provides insights into how an abundant antibiotic found in soil is bound to the enzyme that inactivates it. This work identifies residues for the binding of the antibiotic and probes the contributions of substituting side chains for those in the native protein, providing information regarding hydrophobicity, size, and flexibility of the antibiotic binding site.


Assuntos
Acetiltransferases/metabolismo , Antibacterianos/metabolismo , Bacillus anthracis/enzimologia , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Estreptotricinas/metabolismo , Acetilação , Acetiltransferases/química , Acetiltransferases/genética , Antibacterianos/química , Bacillus anthracis/genética , Bacillus anthracis/metabolismo , Bacillus subtilis/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Estreptotricinas/química
15.
J Vet Sci ; 20(1): 58-62, 2019 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-30541185

RESUMO

Anthrax, caused by Bacillus anthracis, is a non-contagious infectious disease that affects a wide range of animal species (primarily ruminants) including humans. Due to the often-fatal outcome in humans, quick administration of definitely effective antimicrobials is crucial either as prophylaxis or as a clinical case therapy. In this study, 110 B. anthracis strains, temporally, geographically, and genetically different, isolated during anthrax outbreaks in Italy from 1984 to 2017, were screened using a broth microdilution method to determine their susceptibility to 16 clinically relevant antimicrobial agents. The strains were isolated from various matrices (human, animal, and environmental samples) and were representative of thirty distinct genotypes previously identified by 15-loci multiple-locus variable-number of tandem repeats analysis. The antimicrobials tested were gentamicin, ceftriaxone, streptomycin, penicillin G, clindamycin, chloramphenicol, vancomycin, linezolid, cefotaxime, tetracycline, erythromycin, rifampin, amoxicillin, ciprofloxacin, doxycycline, and trimethoprim. All isolates were susceptible to most of the tested antimicrobials, with the exception of trimethoprim for which all of them showed high minimal inhibitory concentration values. An intermediate level of susceptibility was recorded for ceftriaxone and cefotaxime. Although the Centers for Disease Control and Prevention recommend the use of doxycycline, ciprofloxacin, penicillin G, and amoxicillin for treatment of human cases and for post-exposure prophylaxis to anthrax spores, this study shows a high degree of in vitro susceptibility of B. anthracis to many other antimicrobials, suggesting the possibility of an alternative choice for prophylaxis and therapy.


Assuntos
Antibacterianos/farmacologia , Bacillus anthracis/efeitos dos fármacos , Animais , Antraz/tratamento farmacológico , Bacillus anthracis/genética , Bacillus anthracis/fisiologia , Genótipo , Humanos , Itália , Testes de Sensibilidade Microbiana/veterinária , Microbiologia do Solo
16.
PLoS One ; 13(12): e0209120, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30557394

RESUMO

The spore forming pathogen Bacillus anthracis is the etiologic agent of anthrax in humans and animals. It cycles through infected hosts as vegetative cells and is eventually introduced into the environment where it generates an endospore resistant to many harsh conditions. The endospores are subsequently taken up by another host to begin the next cycle. Outbreaks of anthrax occur regularly worldwide in wildlife and livestock, and the potential for human infection exists whenever humans encounter infected animals. It is also possible to encounter intentional releases of anthrax spores, as was the case in October 2001. Consequently, it is important to be able to rapidly establish the provenance of infectious strains of B. anthracis. Here, we compare protein expression in seven low-passage wild isolates and four laboratory strains of B. anthracis grown under identical conditions using LC-MS/MS proteomic analysis. Of the 1,023 total identified proteins, 96 had significant abundance differences between wild and laboratory strains. Of those, 28 proteins directly related to sporulation were upregulated in wild isolates, with expression driven by Spo0A, CodY, and AbrB/ScoC. In addition, we observed evidence of changes in cell division and fatty acid biosynthesis between the two classes of strains, despite being grown under identical experimental conditions. These results suggest wild B. anthracis cells are more highly tuned to sporulate than their laboratory cousins, and this difference should be exploited as a method to differentiate between laboratory and low passage wild strains isolated during an anthrax outbreak. This knowledge should distinguish between intentional releases and exposure to strains in nature, providing a basis for the type of response by public health officials and investigators.


Assuntos
Bacillus anthracis/genética , Bacillus anthracis/fisiologia , Proteínas de Bactérias/genética , Perfilação da Expressão Gênica , Laboratórios , Esporos Bacterianos/fisiologia , Bacillus anthracis/metabolismo , Especificidade da Espécie
17.
Sci Rep ; 8(1): 16108, 2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30382110

RESUMO

Protective antigen (PA) of Bacillus anthracis is being considered as a vaccine candidate against anthrax and its production has been explored in several heterologous host systems. Since the systems tested introduced adverse issues such as inclusion body formation and endotoxin contamination, the production from B. anthracis is considered as a preferred method. The present study examines the effect of PA expression on the metabolism of B. anthracis producing strain, BH500, by comparing it with a control strain carrying an empty plasmid. The strains were grown in a bioreactor and RNA-seq analysis of the producing and non-producing strain was conducted. Among the observed differences, the strain expressing rPA had increased transcription of sigL, the gene encoding RNA polymerase σ54, sigB, the general stress transcription factor gene and its regulators rsbW and rsbV, as well as the global regulatory repressor ctsR. There were also decreased expression of intracellular heat stress related genes such as groL, groES, hslO, dnaJ, and dnaK and increased expression of extracellular chaperons csaA and prsA2. Also, major central metabolism genes belonging to TCA, glycolysis, PPP, and amino acids biosynthesis were up-regulated in the PA-producing strain during the lag phase and down-regulated in the log and late-log phases, which was associated with decreased specific growth rates. The information obtained from this study may guide genetic modification of B. anthracis to improve PA production.


Assuntos
Antígenos de Bactérias/metabolismo , Bacillus anthracis/genética , Toxinas Bacterianas/metabolismo , Transcrição Genética , Antígenos de Bactérias/genética , Bacillus anthracis/crescimento & desenvolvimento , Toxinas Bacterianas/genética , Regulação para Baixo/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Redes e Vias Metabólicas/genética , Análise de Componente Principal , Proteínas Recombinantes/metabolismo , Transcriptoma/genética , Regulação para Cima/genética
18.
mBio ; 9(6)2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30401771

RESUMO

Bacterial spores produced by the Bacillales are composed of concentric shells, each of which contributes to spore function. Spores from all species possess a cortex and coat, but spores from many species possess additional outer layers. The outermost layer of Bacillus anthracis spores, the exosporium, is separated from the coat by a gap known as the interspace. Exosporium and interspace assembly remains largely mysterious. As a result, we have a poor understanding of the overarching mechanisms driving the assembly of one of the most ubiquitous cell types in nature. To elucidate the mechanisms directing exosporium assembly, we generated strains bearing mutations in candidate exosporium-controlling genes and analyzed the effect on exosporium formation. Biochemical and cell biological analyses argue that CotE directs the assembly of CotO into the spore and that CotO might be located at or close to the interior side of the cap. Taken together with data showing that CotE and CotO interact directly in vitro, we propose a model in which CotE and CotO are important components of a protein interaction network that connects the exosporium to the forespore during cap formation and exosporium elongation. Our data also suggest that the cap interferes with coat assembly at one pole of the spore, altering the pattern of coat deposition compared to the model organism Bacillus subtilis We propose that the difference in coat assembly patterns between these two species is due to an inherent flexibility in coat assembly, which may facilitate the evolution of spore outer layer complexity.IMPORTANCE This work dramatically improves our understanding of the assembly of the outermost layer of the B. anthracis spore, the exosporium, a layer that encases spores from many bacterial species and likely plays important roles in the spore's interactions with the environment, including host tissues. Nonetheless, the mechanisms directing exosporium assembly into a shell surrounding the spore are still very poorly understood. In this study, we clarify these mechanisms by the identification of a novel protein interaction network that directs assembly to initiate at a specific subcellular location in the developing cell. Our results further suggest that the presence or absence of an exosporium has a major impact on the assembly of other more interior spore layers, thereby potentially explaining long-noted differences in spore assembly between B. anthracis and the model organism B. subtilis.


Assuntos
Bacillus anthracis/fisiologia , Proteínas de Bactérias/metabolismo , Esporos Bacterianos/fisiologia , Bacillus anthracis/genética , Bacillus subtilis/genética , Bacillus subtilis/fisiologia , Proteínas de Bactérias/genética , Parede Celular/metabolismo , Mutação , Mapas de Interação de Proteínas , Esporos Bacterianos/genética
19.
mBio ; 9(6)2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30401780

RESUMO

Bacillus anthracis is a Gram-positive bacillus that under conditions of environmental stress, such as low nutrients, can convert from a vegetative bacillus to a highly durable spore that enables long-term survival. The sporulation process is regulated by a sequential cascade of dedicated transcription factors but requires key nutrients to complete, one of which is iron. Iron acquisition by the iron-scavenging siderophore petrobactin is required for vegetative growth of B. anthracis under iron-depleted conditions and in the host. However, the extent to which petrobactin is involved in spore formation is unknown. This work shows that efficient in vitro sporulation of B. anthracis requires petrobactin, that the petrobactin biosynthesis operon (asbA to -F) is induced prior to sporulation, and that the siderophore itself associates with spores. Petrobactin is also required for oxidative stress protection during late-stage growth and for wild-type levels of sporulation in sporulation medium. Sporulation in bovine blood was found to be petrobactin dependent. Collectively, the in vitro contributions of petrobactin to sporulation as well as growth imply that petrobactin may be required for B. anthracis transmission via the spore during natural infections, in addition to its key known functions during active anthrax infections.IMPORTANCE Bacillus anthracis causes the disease anthrax, which is transmitted via its dormant, spore phase. However, conversion from bacillus to spore is a complex, energetically costly process that requires many nutrients, including iron. B. anthracis requires the siderophore petrobactin to scavenge iron from host environments. We show that, in the Sterne strain, petrobactin is required for efficient sporulation, even when ample iron is available. The petrobactin biosynthesis operon is expressed during sporulation, and petrobactin is biosynthesized during growth in high-iron sporulation medium, but instead of being exported, the petrobactin remains intracellular to protect against oxidative stress and improve sporulation. It is also required for full growth and sporulation in blood (bovine), an essential step for anthrax transmission between mammalian hosts.


Assuntos
Bacillus anthracis/crescimento & desenvolvimento , Benzamidas/metabolismo , Estresse Oxidativo , Esporos Bacterianos/crescimento & desenvolvimento , Animais , Bacillus anthracis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bovinos , Ferro/metabolismo , Óperon , Sideróforos/genética , Sideróforos/metabolismo
20.
PLoS Negl Trop Dis ; 12(11): e0006908, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30418972

RESUMO

Anthrax is a global re-emerging zoonotic disease and is an endemic disease in China, especially in rural regions. In this study, the general characteristics of human anthrax outbreaks that occurred in areas of northwestern China over the past decade have been described. Meanwhile, the genetic characteristics of Bacillus anthracis isolated from these areas from 1990 to 2016 were analyzed by means of canonical single-nucleotide polymorphism (canSNP) analysis and multilocus variable-number tandem repeat analysis (MLVA) with 15 markers. Five sublineages/subgroups, namely, A.Br.001/002, A.Br.Vollum, A.Br.Aust94, A.Br.Ames and A.Br.008/009, were detected by using 13 canSNP sites. All of the sublineages were found in Xinjiang province, while one sublineage was found in Shaanxi, two in Gansu, three in Qinghai and four in Inner Mongolia. However, the geographical distribution of the B. anthracis populations exhibited different canSNP characteristics from those of the strains isolated before 1990 in China. In contrast to previous data, the A.Br.Ames subgroup was also observed to be scattered from Inner Mongolia to other provinces. All 106 strains were assigned to 36 MLVA15 genotypes, and 21 of these types were first observed in this study. The strains collected from anthrax outbreaks in recent decade were classified as subgroups A.Br.001/002 and A.Br.Ames and identified as genotypes MLVA15-28, MLVA15-30, MLVA15-31, MLVA15-38, MLVA15-CHN3, and MLVA15-CHN18. By canSNP analysis and MLVA, we found that the diversification of MLVA genotypes and the geographical distribution of B. anthracis populations is gradually becoming balanced across northwestern China. This study also provides preliminary survey results regarding the population diversity of B. anthracis in China, which will help promote the prevention and control of this important disease.


Assuntos
Antraz/epidemiologia , Bacillus anthracis/genética , Bacillus anthracis/isolamento & purificação , Animais , Antraz/microbiologia , Antraz/prevenção & controle , Antraz/transmissão , Bacillus anthracis/classificação , Bovinos , China/epidemiologia , Surtos de Doenças , Equidae , Variação Genética , Genótipo , Humanos , Gado , Repetições Minissatélites , Mongólia/epidemiologia , Tipagem de Sequências Multilocus , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNA , Ovinos , Zoonoses/epidemiologia , Zoonoses/prevenção & controle , Zoonoses/transmissão
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