Disruption of SpoIIID decreases sporulation, increases extracellular proteolytic activity and virulence in Bacillus anthracis.

Endospores are important for maintenance of the B. anthracis lifecycle and necessary for its effective spread between hosts. Our experiments with B. anthracis showed that disruption of SpoIIID results in a spore formation defect, as determined by heat resistance assays and microscopic assessment. We further found complete engulfment by the ΔspoIIID mutant strain by membrane morphology staining but no synthesis of the clarity coat and exosporium by transmission electron microscopy. Reduced transcription and expression of small acid-soluble spore protein(sasP-2) and the spore development associated genes (σK, gerE and cotE) in the mother cell were found in the ΔspoIIID strain, suggesting that the spore formation defect in B. anthracis A16R is related to decreased transcription and expression of these genes. Extracellular protease and virulence enhancement in the ΔspoIIID strain may be related to the elevation of metalloproteinases (TasA and Camelysin) levels. Our findings pave the way for further research on the regulation network of sporulation, survival and virulence in these two morphological forms of B. anthracis.

Genome sequence of Bacillus anthracis typing phage AP631.

AP631, a virulent bacteriophage of Bacillus anthracis, is widely used in China to identify anthrax bacteria. In this study, we report the complete AP631 phage genome sequence as well as comparative genomic analysis with other bacteriophages of B. cereus and related species. The double-stranded circular DNA genome of phage AP631 was 39,549 bp in length with 35.01% G + C content. The phage genome contained 56 putative protein-coding genes but no rRNA or tRNA genes. Comparative phylogenetic analysis of the phage major capsid proteins and terminase large subunits showed that phage AP631 belongs to the B. cereus sensu lato phage clade II. Comparative genomic analysis revealed a high degree of sequence similarity between phage AP631 and B. anthracis phages Wbeta, Gamma, Cherry, and Fah, as well as three AP631-specific genes bearing no significant similarity to those of other phages.

Analysis of Soluble protein complexes in Shigella flexneri reveals the influence of temperature on the amount of lipopolysaccharide.

Shigella flexneri, which is closely related to Escherichia coli, is the most common cause of the endemic form of shigellosis. In this study, 53 homomultimeric protein complexes and nine heteromultimeric protein complexes from S. flexneri 2a strain 2457T were separated and identified. Among these, three potential homomultimeric protein complexes had not been previously described. One complex, thought to be composed of 12 PhoN1 subunits, is a periplasmic protein with an unknown physiological role encoded on the virulence plasmid of S. flexneri. The abundance of the protein complexes was compared following growth at 37 or 30°C, and the abundance of three protein complexes (PyrB-PyrI, GlmS, and MglB) related to the synthesis of lipopolysaccharides (LPS) appeared to be temperature-dependent. Many studies have shown that LPS is essential to the virulence of S. flexneri. Here, we report the influence of temperature on the amount of LPS.

The effect of quorum sensing system for growth competitiveness on Shigella flexneri.

Quorum sensing (QS) regulates the onset of bacterial social responses related to cell density. Comparison between the gene sequences of all components of QS system of Escherichia coli and Shigella strains, shows that the QS system is generally lost or mutated in Shigella. Since AI-2 is produced and processed by the lsr operon, we analyzed the potential function of the lsr operon. We first detected AI-2 in Shigella flexneri 2a strain 301 through the reporter bacteria Vibrio harveyi BB170, indicating that S. flexneri can produce AI-2. Then, the lsr operon of E. coli MG1655 was cloned into S. flexneri using the Golden Gate method. Colony counting experiments showed that the QS system recovery strain had growth advantage over the wild-type strain when they were mixed and cultured. The preliminary comparative proteomics analysis showed that the lsr operon could be expressed and the abundance of stress response proteins also changed when the QS system was introduced into S. flexneri.

Preparation and immunogenicity-evaluation of typhoid O-specific polysaccharides bio-conjugate vaccines.

Typhoid fever caused by Salmonella Typhi is still a major public health problem in developing countries. In this study, we constructed a genetically modified Salmonella Typhi strain expressing O-specific polysaccharides (OPS) antigen conjugated to a carrier, recombinant Pseudomonas aeruginosa exotoxin A(rEPA N29). The conjugates (OPS-rEPA N29) were further purified and evaluated for their immunogenicity. The results of ELISA showed that the conjugates evoked higher titers of IgG than OPS, suggesting that rEPAN29 increased immunogenicity of OPS significantly as a carrier. Moreover, three injections with 3-week interval evoked slightly higher titers of IgG than three injections with 2-week interval. However, injection of excess conjugates could not evoke higher titers of IgG against lipid polysaccharide (LPS). In summary, our study provides a new strategy for preparing polysaccharides-protein conjugate vaccines as well as similar bio-conjugate vaccines of other Gram-negative pathogens.

The function of PlcR in Bacillus anthracis vaccine strain A16R.

Bacillus anthracis, B. thuringiensis and B. cereus are members of the B. cereus group. They share high genetic similarity. Whereas plcR (Phospholipase C regulator) usually encodes a functional pleiotropic activator protein in B. cereus and B. thuringiensis isolates, a characteristic nonsense mutation is found in all B. anthracis strains investigated, making the gene dysfunctional. To study the function of PlcR in B. anthracis, we used the B. cereus CMCC63301 genome as a template and constructed a recombinant expression plasmid pBE2A-plcR, and introduced it into the B. anthracis vaccine strain A16R, and then analyzed the activity of the hemolysin and sphingomyelinase. The results showed that transformation of B. anthracis with plasmid pBE2A-plcR carrying the native B. cereus plcR gene active the expression of sphingomyelinase gene, but did not activate expression of hemolysin genes of B. anthracis A16R.

[Clustered regularly interspaced short palindromic repeats (CRISPR) site in Bacillus anthracis].

OBJECTIVE: To investigate the polymorphism of clustered regularly interspaced short palindromic repeats (CRISPR) in Bacillu santhracis and the application to molecular typing based on the polymorphism of CRISPR in B. anthracis. METHODS: We downloaded the whole genome sequence of 6 B. anthracis strains and extracted the CRISPR sites. We designed the primers of CRISPR sites and amplified the CRISPR fragments in 193 B. anthracis strains by PCR and sequenced these fragments. In order to reveal the polymorphism of CRISPR in B. anthracis, wealigned all the extracted sequences and sequenced results by local blasting. At the same time, we also analyzed the CRISPR sites in B. cereus and B. thuringiensis. RESULTS: We did not find any polymorphism of CRISPR in B. anthracis. CONCLUSION: The molecular typing approach based on CRISPR polymorphism is not suitable for B. anthracis, but it is possible for us to distinguish B. anthracis from B. cereus and B. thuringiensis.

[Screening and identification of glycoproteins from Campylobacter jejuni NCTC 11168 based on specific affinity between lectins and glycoproteins].

OBJECTIVE: To screen glycoproteins from Campylobacter jejuni NCTC 11168. METHODS: We introduced a screening strategy based on specific affinity between lectins and glycoproteins. First, the specific affinity between Soybean Agglutinin (SBA) and glycoproteins in C. jejuni was confirmed by western blotting. Then, magnetic beads coating with Soybean Agglutinin (SBA) were used to capture the putative glycoproteins. Putative glycoproteins were competitively eluted by N-acetylgalactosamine and separated by two-dimensional gel electrophoresis. Spots in gels were further analyzed by mass spectrometry. RESULTS: A total of 22 proteins were identified by mass spectrometric analysis, of which 5 have ever been identified to be glycoproteins. Others including Cj0633 have not been proved to be glycosylated so far. CONCLUSION: The method could be used to screen glycoproteins in bacteria.

Genome Sequence and Phenotypic Analysis of a Protein Lysis-Negative, Attenuated Anthrax Vaccine Strain.

Bacillus anthracis is a Gram-positive bacterium that causes the zoonotic disease anthrax. Here, we studied the characteristic phenotype and virulence attenuation of the putative No. II vaccine strain, PNO2, which was reportedly introduced from the Pasteur Institute in 1934. Characterization of the strain showed that, compared with the control strain, A16Q1, the attenuated PNO2 (PNO2D1) was phospholipase-positive, with impaired protein hydrolysis and significantly reduced sporulation. Additionally, PNO2D1 significantly extended the survival times of anthrax-challenged mice. An evolutionary tree analysis revealed that PNO2D1 was not a Pasteur strain but was more closely related to a Tsiankovskii strain. A database comparison revealed a seven-base insertion mutation in the nprR gene. Although it did not block nprR transcription, the insertion mutation resulted in the premature termination of protein translation. nprR deletion of A16Q1 resulted in a nonproteolytic phenotype that could not sporulate. The database comparison revealed that the abs gene is also prone to mutation, and the abs promoter activity was much lower in PNO2D1 than in A16Q1. Low abs expression may be an important reason for the decreased virulence of PNO2D1.

The proteome of Shigella flexneri 2a 2457T grown at 30 and 37 degrees C.

To upgrade the proteome reference map of Shigella flexneri 2a 2457T, the protein expression profiles of log phase and stationary phase cells grown at 30 and 37 degrees C were thoroughly analyzed using multiple overlapping narrow pH range (between pH 4.0 and 11.0) two-dimensional gel electrophoresis. A total of 723 spots representing 574 protein entries were identified by MALDI-TOF/TOF MS, including the majority of known key virulence factors. 64 hypothetical proteins and six misannotated proteins were also experimentally identified. A comparison between the four proteome maps showed that most of the virulence-related proteins were up-regulated at 37 degrees C, and the differences were more notable in stationary phase cells, suggesting that the expressions of these virulence factors were not only controlled by temperature but also controlled by the nutrients available in the environment. The expression patterns of some virulence-related genes under the four different conditions suggested that they might also be regulated at the post-transcriptional level. A further significant finding was that the expression of the protein ArgT was dramatically up-regulated at 30 degrees C. The results of semiquantitative RT-PCR analysis showed that expression of argT was not regulated at the transcriptional level. Therefore, we carried out a series of experiments to uncover the mechanism regulating ArgT levels and found that the differential expression of ArgT was due to its degradation by a periplasmic protease, HtrA, whose activity, but not its synthesis, was affected by temperature. The cleavage site in ArgT was between position 160 (Val) and position 161 (Ala). These results may provide useful insights for understanding the physiology and pathogenesis of S. flexneri.

Orthogonal modular biosynthesis of nanoscale conjugate vaccines for vaccination against infection.

Conjugate vaccines represent one of the most effective means for controlling the occurrence of bacterial diseases. Although nanotechnology has been greatly applied in the field of vaccines, it is seldom used for conjugate vaccine research because it is very difficult to connect polysaccharides and nanocarriers. In this work, an orthogonal and modular biosynthesis method was used to produce nanoconjugate vaccines using the SpyTag/SpyCatcher system. When SpyTag/SpyCatcher system is combined with protein glycosylation technology, bacterial O-polysaccharide obtained from Shigela flexneri 2a can be conjugated onto the surfaces of different virus-like particles (VLPs) in a biocompatible and controlled manner. After confirming the excellent lymph node targeting and humoral immune activation abilities, these nanoconjugate vaccines further induced efficient prophylactic effects against infection in a mouse model. These results demonstrated that natural polysaccharide antigens can be easily connected to VLPs to prepare highly efficient nanoconjugate vaccines. To the best of the researchers' knowledge, this is the first time VLP-based nanoconjugate vaccines are produced efficiently, and this strategy could be applied to develop various pathogenic nanoconjugate vaccines. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material (Figs. S1-S9) is available in the online version of this article at 10.1007/s12274-021-3713-4.

Rapid Identification of Bacillus anthracis In Silico and On-Site Using Novel Single-Nucleotide Polymorphisms.

Bacillus anthracis is a spore-forming bacterium that causes life-threatening infections in animals and humans and has been used as a bioterror agent. Rapid and reliable detection and identification of B. anthracis are of primary interest for both medical and biological threat-surveillance purposes. Few chromosomal sequences provide enough polymorphisms to clearly distinguish B. anthracis from closely related species. We analyzed 18 loci of the chromosome of B. anthracis and discovered eight novel single-nucleotide polymorphism (SNP) sites that can be used for the specific identification of B. anthracis. Using these SNP sites, we developed software-named AGILE V1.1 (anthracis genome-based identification with high-fidelity E-probe)-for easy, user-friendly identification of B. anthracis from whole-genome sequences. We also developed a recombinase polymerase amplification-Cas12a-based method that uses nucleic acid extracts for the specific, rapid, in-the-field identification of B. anthracis based on these SNPs. Via this method and B. anthracis-specific CRISPR RNAs for the target CR5_2, CR5_1, and Ba813 SNPs, we clearly detected 5 aM genomic DNA. This study provides two simple and reliable methods suitable for use in local hospitals and public health programs for the detection of B. anthracis. IMPORTANCE Bacillus anthracis is the etiologic agent of anthrax, a fatal disease and a potential biothreat. A specific, accurate, and rapid method is urgently required for the identification of B. anthracis. We demonstrate the potential of using eight novel SNPs for the rapid and accurate detection of B. anthracis via in silico and laboratory-based testing methods. Our findings have important implications for public health responses to disease outbreaks and bioterrorism threats.

A CRISPR/Cas12a-based DNAzyme visualization system for rapid, non-electrically dependent detection of Bacillus anthracis.

As next-generation pathogen detection methods, CRISPR-Cas-based detection methods can perform single-nucleotide polymorphism (SNP) level detection with high sensitivity and good specificity. They do not require any particular equipment, which opens up new possibilities for the accurate detection and identification of Bacillus anthracis. In this study, we developed a complete detection system for B. anthracis based on Cas12a. We used two chromosomally located SNP targets and two plasmid targets to identify B. anthracis with high accuracy. The CR5 target is completely new. The entire detection process can be completed within 90 min without electrical power and with single-copy level sensitivity. We also developed an unaided-eye visualization system based on G4-DNAzyme for use with our CRISPR-Cas12a detection system. This visualization system has good prospects for deployment in field-based point-of-care detection. We used the antisense nucleic acid CatG4R as the detection probe, which showed stronger resistance to interference from components of the solution. CatG4R can also be designed as an RNA molecule for adaptation to Cas13a detection, thereby broadening the scope of the detection system.

Safety and immunogenicity of a new glycoengineered vaccine against Acinetobacter baumannii in mice.

Acinetobacter baumannii poses a serious threat to human health, mainly because of its widespread distribution and severe drug resistance. However, no licensed vaccines exist for this pathogen. In this study, we created a conjugate vaccine against A. baumannii by introducing an O-linked glycosylation system into the host strain. After demonstrating the ability of the vaccine to elicit Th1 and Th2 immune responses and observing its good safety in mouse a model, the strong in vitro bactericidal activity and prophylactic effects of the conjugate vaccine against infection were further demonstrated by evaluating post-infection tissue bacterial loads, observing suppressed serum pro-inflammatory cytokine levels. Additionally, the broad protection from the vaccine was further proved via lethal challenge with A. baumannii. Overall, these results indicated that the conjugate vaccine could elicit an efficient immune response and provide good protection against A. baumannii infection in murine sepsis models. Thus, the conjugate vaccine can be considered as a promising candidate vaccine for preventing A. baumannii infection.

Curing the plasmid pXO2 from Bacillus anthracis A16 using plasmid incompatibility.

Plasmid incompatibility, which has no effect on other plasmids or chromosomal genes, can be used to cure a target plasmid. In this report, we successfully cured the plasmid pXO2 from Bacillus anthracis A16 with a newly constructed, incompatible plasmid pKSV7-oriIV and obtained a new pXO2-cured strain, designated A16PI2. This is the first time that a plasmid was cured from the B. anthracis wild-type strain A16 utilizing this principle, which could be considered as an efficacious method to cure large plasmids.

Expression of Bordetella pertussis Antigens Fused to Different Vectors and Their Effectiveness as Vaccines.

Pertussis is an acute respiratory tract infection caused by Bordetella pertussis. Even though its current vaccine coverage is relatively broad, they still have some shortcomings such as short protection time and might be incapable of blocking the spread of the disease. In this study, we developed new pertussis vaccine candidates by separately fusing three pertussis antigens (B. pertussis fimbriae 2 "Fim2", pertussis toxin S1 subunit "PtxS1", and filamentous hemagglutinin "FHA1877-2250") to each of two immune-boosting carrier proteins (B subunits of AB5 toxin family: cholera toxin B subunit "CTB" and shiga toxin B subunit "StxB"). We then immunized mice with these fusion antigens and found that they significantly increased the serum antibody titers and elicited high bactericidal activity against B. pertussis. After CTB-or StxB-fused antigen-immunized mice were challenged with a non-lethal dose of B. pertussis, the bacterial loads in different tissues of these mice were significantly reduced, and their lung damage was nearly invisible. Furthermore, we also demonstrated that these candidate vaccines could provide strong prophylactic effects against a lethal challenge with B. pertussis. Overall, our candidate vaccines conferred better immune protection to mice compared with pertussis antigen alone. This B5 subunit-based vaccine strategy provides a promising option for vaccine design.

Application of CRISPR/Cas9 System for Plasmid Elimination and Bacterial Killing of Bacillus cereus Group Strains.

The CRISPR-Cas system has been widely applied in prokaryotic genome editing with its high efficiency and easy operation. We constructed some "scissors plasmids" via using the temperature-sensitive pJOE8999 shuttle plasmid, which carry the different 20nt (N20) guiding the Cas9 nuclease as a scissors to break the target DNA. We successfully used scissors plasmids to eliminate native plasmids from Bacillus anthracis and Bacillus cereus, and specifically killed B. anthracis. When curing pXO1 and pXO2 virulence plasmids from B. anthracis A16PI2 and A16Q1, respectively, we found that the plasmid elimination percentage was slightly higher when the sgRNA targeted the replication initiation region (96-100%), rather than the non-replication initiation region (88-92%). We also tried using a mixture of two scissors plasmids to simultaneously eliminate pXO1 and pXO2 plasmids from B. anthracis, and the single and double plasmid-cured rates were 29 and 14%, respectively. To our surprise, when we used the scissor plasmid containing two tandem sgRNAs to cure the target plasmids pXO1 and pXO2 from wild strain B. anthracis A16 simultaneously, only the second sgRNA could guide Cas9 to cleave the target plasmid with high efficiency, while the first sgRNA didn't work in all the experiments we designed. When we used the CRISPR/cas9 system to eliminate the pCE1 mega-virulence plasmid from B. cereus BC307 by simply changing the sgRNA, we also obtained a plasmid-cured isogenic strain at a very high elimination rate (69%). The sterilization efficiency of B. anthracis was about 93%, which is similar to the efficiency of plasmid curing, and there was no significant difference in the efficiency of among the scissors plasmids containing single sgRNA, targeting multi-sites, or single-site targeting and the two tandem sgRNA. This simple and effective curing method, which is applicable to B. cereus group strains, provides a new way to study these bacteria and their virulence profiles.

Obtaining Specific Sequence Tags for Yersinia pestis and Visually Detecting Them Using the CRISPR-Cas12a System.

Three worldwide historical plague pandemics resulted in millions of deaths. Yersinia pestis, the etiologic agent of plague, is also a potential bioterrorist weapon. Simple, rapid, and specific detection of Y. pestis is important to prevent and control plague. However, the high similarity between Y. pestis and its sister species within the same genus makes detection work problematic. Here, the genome sequence from the Y. pestis CO92 strain was electronically separated into millions of fragments. These fragments were analyzed and compared with the genome sequences of 539 Y. pestis strains and 572 strains of 20 species within the Yersinia genus. Altogether, 97 Y. pestis-specific tags containing two or more single nucleotide polymorphism sites were screened out. These 97 tags efficiently distinguished Y. pestis from all other closely related species. We chose four of these tags to design a Cas12a-based detection system. PCR-fluorescence methodology was used to test the specificity of these tags, and the results showed that the fluorescence intensity produced by Y. pestis was significantly higher than that of non-Y. pestis (p < 0.0001). We then employed recombinase polymerase amplification and lateral flow dipsticks to visualize the results. Our newly developed plasmid-independent, species-specific library of tags completely and effectively screened chromosomal sequences. The detection limit of our four-tag Cas12a system reached picogram levels.

Global analysis of a plasmid-cured Shigella flexneri strain: new insights into the interaction between the chromosome and a virulence plasmid.

Shigella flexneri is an important human pathogen that causes dysentery, and remains a significant threat to public health, particularly in developing countries. The virulence of this pathogen is dependent on an acquired virulence plasmid. To investigate the crosstalk between the bacterial chromosome and the exogenous virulence plasmid, a virulence plasmid-cured strain was constructed using plasmid incompatibility. The global patterns of gene expression of this strain compared with the wild-type strain were analyzed using 2-DE combined with MALDI-TOF MS. Most known virulence factors of S. flexneri were identified in the 2-DE gels. Interestingly, the expression of the glycerol 3-phosphate (glp) regulon-encoded proteins was increased when the virulence plasmid was absent. Microarray analysis confirmed that regulation occurred at the transcriptional level. Purification and identification of DNA binding proteins with affinity for the regulatory region of the glp genes revealed that regulation mediated by the virulence plasmid to control the expression of the glp regulon might in turn be mediated by protein GlpR. To our knowledge, this is the first study analyzing the interaction between a pathogen chromosome and a virulence plasmid at the proteomic level.

A novel anti-virulence gene revealed by proteomic analysis in Shigella flexneri 2a.

BACKGROUND: Shigella flexneri is a gram-negative, facultative pathogen that causes the majority of communicable bacterial dysenteries in developing countries. The virulence factors of S. flexneri have been shown to be produced at 37 degrees C but not at 30 degrees C. To discover potential, novel virulence-related proteins of S. flexneri, we performed differential in-gel electrophoresis (DIGE) analysis to measure changes in the expression profile that are induced by a temperature increase. RESULTS: The ArgT protein was dramatically down-regulated at 37 degrees C. In contrast, the ArgT from the non-pathogenic E. coli did not show this differential expression as in S. flexneri, which suggested that argT might be a potential anti-virulence gene. Competitive invasion assays in HeLa cells and in BALB/c mice with argT mutants were performed, and the results indicated that the over-expression of ArgTY225D would attenuate the virulence of S. flexneri. A comparative proteomic analysis was subsequently performed to investigate the effects of ArgT in S. flexneri at the molecular level. We show that HtrA is differentially expressed among different derivative strains. CONCLUSION: Gene argT is a novel anti-virulence gene that may interfere with the virulence of S. flexneri via the transport of specific amino acids or by affecting the expression of the virulence factor, HtrA.