YPTB3816 of 'Yersinia pseudotuberculosis' strain IP32953 is a virulence-related metallo-oligopeptidase

Background: although bacterial peptidases are known to be produced by various microorganisms, including pathogenic bacteria, their role in bacterial physiology is not fully understood. In particular, oligopeptidases are thought to be mainly involved in degradation of short peptides e.g. leader peptides released during classical protein secretion pathways. The aim of this study was to investigate effects of inactivation of an oligopeptidase encoding gene opdA gene of Yersinia pseudotuberculosis on bacterial properties in vivo and in vitro, and to test dependence of the enzymatic activity of the respective purified enzyme on the presence of different divalent cations. Results: in this study we found that oligopeptidase OpdA of Yersinia pseudotuberculosis is required for bacterial virulence, whilst knocking out the respective gene did not have any effect on bacterial viability or growth rate in vitro. In addition, we studied enzymatic properties of this enzyme after expression and purification from E. coli. Using an enzyme depleted of contaminant divalent cations and different types of fluorescently labelled substrates, we found strong dependence of its activity on the presence of particular cations. Unexpectedly, Zn2+ showed stimulatory activity only at low concentrations, but inhibited the enzyme at higher concentrations. In contrast, Co2+, Ca2+ and Mn2+ stimulated activity at all concentrations tested, whilst Mg2+ revealed no effect on the enzyme activity at all concentrations used. Conclusions: the results of this study provide valuable contribution to the investigation of bacterial peptidases in general, and that of metallo-oligopeptidases in particular. This is the first study demonstrating that opdA in Yersinia pseudotuberculsosis is required for pathogenicity. The data reported are important for better understanding of the role of OpdA-like enzymes in pathogenesis in bacterial infections. Characterisation of this protein may serve as a basis for the development of novel antibacterials based on specific inhibition of this peptidase activity

Biochemical studies on Francisella tularensis RelA in (p)ppGpp Biosynthesis

2 ABSTRACT The bacterial stringent response is induced by nutrient deprivation and is mediated by enzymes of the RSH superfamily that control concentrations of the "alarmones" (p)ppGpp. This regulatory pathway is present in the vast majority of pathogens and has been proposed as a potential antibacterial target. Current understanding of RelA mediated responses are based on biochemical studies using Escherichia coli as a model. In comparison, the Francisella tularensis RelA sequence contains a truncated regulatory C-terminal region and an unusual synthetase motif (EXSD). Biochemical analysis of Francisella tularensis RelA showed the similarities and differences of this enzyme compared to the model RelA from Escherichia coli. Purification of the enzyme yielded a stable dimer capable of reaching concentrations of 10 mg/mL. In contrast to other enzymes from the RelA/SpoT homologue superfamily, activity assays with F. tularensis RelA demonstrate a high degree of specificity for GTP as a pyrophosphate acceptor, with no measurable turnover for GDP. Steady state kinetic analysis of F. tularensis RelA gave saturation activity curves that best fitted a sigmoidal function. This kinetic profile can result from allosteric regulation and further measurements with potential allosteric regulators demonstrated activation by ppGpp with an EC 50 of 60 ± 1.9 μM. Activation of F. tularensis RelA by stalled ribosomal complexes formed with ribosomes purified from Escherichia coli MRE600 was observed, but interestingly, significantly weaker activation with ribosomes isolated from Francisella philomiragia

Synthetic routes, characterization and photophysical properties of luminescent, surface functionalized nanodiamonds

The functionalization of small diameter (ca. 50 nm) polycarboxylated nanodiamond particles using amide coupling methodologies in both water and acetonitrile solvent has been investigated. In this manner, the surfaces of nanodiamond particles were adorned with different luminescent moieties, including a green fluorescent 1,8-naphthalimide species (Nap-1), and a red emitting ruthenium(II) tris-bipyridine complex (Ru-1), as well as dual functionalization with both luminophores. Comprehensive characterization of the surface functionalized nanodiamonds has been achieved using a combination of dynamic light scattering, nanoparticle tracking analysis, transmission electron microscopy, X-ray photoelectron spectroscopy, zeta potential measurements, microwave plasma atomic emission spectroscopy and time-resolved photophysics. The tendency of the functionalized nanodiamonds to aggregate reflects the degree of surface substitution, yielding small aggregates with typical particle sizes ca. 150 nm. This is likely to be driven by the reduction of the zeta potential, concomitant with the conversion of surface charged carboxylate groups to neutral amide functions. The results show that luminescent nanodiamond materials can be synthesised with tuneable photophysical properties

The influence of extrachromosomal elements in the anthrax “cross-over” strain Bacillus cereus G9241

Bacillus cereus G9241 was isolated from a welder who survived a pulmonary anthrax-like disease. Strain G9241 carries two virulence plasmids, pBCX01 and pBC210, as well as an extrachromosomal prophage, pBFH_1. pBCX01 has 99.6% sequence identity to pXO1 carried by Bacillus anthracis and encodes the tripartite anthrax toxin genes and atxA, a mammalian virulence transcriptional regulator. This work looks at how the presence of pBCX01 and temperature may affect the lifestyle of B. cereus G9241 using a transcriptomic analysis and by studying spore formation, an important part of the B. anthracis lifecycle. Here we report that pBCX01 has a stronger effect on gene transcription at the mammalian infection relevant temperature of 37°C in comparison to 25°C. At 37°C, the presence of pBCX01 appears to have a negative effect on genes involved in cell metabolism, including biosynthesis of amino acids, whilst positively affecting the transcription of many transmembrane proteins. The study of spore formation showed B. cereus G9241 sporulated rapidly in comparison to the B. cereus sensu stricto type strain ATCC 14579, particularly at 37°C. The carriage of pBCX01 did not affect this phenotype suggesting that other genetic elements were driving rapid sporulation. An unexpected finding of this study was that pBFH_1 is highly expressed at 37°C in comparison to 25°C and pBFH_1 expression leads to the production of Siphoviridae-like phage particles in the supernatant of B. cereus G9241. This study provides an insight on how the extrachromosomal genetic elements in B. cereus G9241 has an influence in bacterial phenotypes

An integrated computational-experimental approach reveals Yersinia pestis genes essential across a narrow or a broad range of environmental conditions

Background The World Health Organization has categorized plague as a re-emerging disease and the potential for Yersinia pestis to also be used as a bioweapon makes the identification of new drug targets against this pathogen a priority. Environmental temperature is a key signal which regulates virulence of the bacterium. The bacterium normally grows outside the human host at 28 °C. Therefore, understanding the mechanisms that the bacterium used to adapt to a mammalian host at 37 °C is central to the development of vaccines or drugs for the prevention or treatment of human disease. Results Using a library of over 1 million Y. pestis CO92 random mutants and transposon-directed insertion site sequencing, we identified 530 essential genes when the bacteria were cultured at 28 °C. When the library of mutants was subsequently cultured at 37 °C we identified 19 genes that were essential at 37 °C but not at 28 °C, including genes which encode proteins that play a role in enabling functioning of the type III secretion and in DNA replication and maintenance. Using genome-scale metabolic network reconstruction we showed that growth conditions profoundly influence the physiology of the bacterium, and by combining computational and experimental approaches we were able to identify 54 genes that are essential under a broad range of conditions. Conclusions Using an integrated computational-experimental approach we identify genes which are required for growth at 37 °C and under a broad range of environments may be the best targets for the development of new interventions to prevent or treat plague in humans

From cereus to anthrax and back again : the role of the PlcR regulator in the “cross-over” strain Bacillus cereus G9241

The plcR gene, which encodes the pleiotropic transcriptional regulator of secreted proteins found in most members of the Bacillus cereus group, is truncated in all Bacillus anthracis isolates. The current dogma suggests this truncation was evolved to accommodate the acquisition of the anthrax toxin regulator, AtxA. However, the B. cereus-B. anthracis “cross-over” strain Bacillus cereus G9241, isolated from a Louisiana welder suffering from an anthrax-like infection, appears to contradict the proposed dogma as it encodes intact copies of both regulators. Here we report that when cultured at 25 °C, cell free B. cereus G9241 culture supernatants are cytotoxic and haemolytic to various eukaryotic cells in addition to insect haemocytes from Manduca sexta. However, this cytotoxic and haemolytic activity of the culture supernatant is lost when the bacteria are grown at 37 °C, behaving much like the supernatants generated by B. anthracis. Using a combination of genetic and proteomic approaches, we identified several PlcR-regulated toxins secreted at 25 °C. We demonstrate that a limiting step for the production of these virulence factors at 37 °C exists within the PlcR-PapR regulation circuit in strain G9241, giving rise to the temperature-dependent haemolytic and cytotoxic activity of the culture supernatants. Differential expression of the protease responsible in processing the PlcR quorum sensing activator PapR appears to be responsible for this phenotype. This study confirms that B. cereus G9241 is able to ‘switch’ between B. cereus and B. anthracis–like phenotypes in a temperature-dependent manner, potentially accommodating the activities of both PlcR and AtxA

From cereus to anthrax and back again: Assessment of the temperature-dependent phenotypic switching in the "cross-over" strain Bacillus cereus G9241

Bacillus cereus G9241 was isolated from a Louisiana welder suffering from an anthrax-like infection. The organism carries two transcriptional regulators that have previously been proposed to be incompatible with each other in Bacillus anthracis: the pleiotropic transcriptional regulator PlcR found in most members of the Bacillus cereus group but truncated in all B. anthracis isolates, and the anthrax toxin regulator AtxA found in all B. anthracis strains and a few B. cereus sensu stricto strains. Here we report cytotoxic and hemolytic activity of cell free B. cereus G9241 culture supernatants cultured at 25°C to various eukaryotic cells. However, this is not observed at the mammalian infection relevant temperature 37°C, behaving much like the supernatants generated by B. anthracis. Using a combination of genetic and proteomic approaches to understand this unique phenotype, we identified several PlcR-regulated toxins to be secreted highly at 25°C compared to 37°C. Furthermore, results suggest that differential expression of the protease involved in processing the PlcR quorum sensing activator molecule PapR appears to be the limiting step for the production of PlcR-regulated toxins at 37°C, giving rise to the temperature-dependent hemolytic and cytotoxic activity of the culture supernatants. This study provides an insight on how B. cereus G9241 is able to “switch” between B. cereus and B. anthracis–like phenotypes in a temperature-dependent manner, potentially accommodating the activities of both PlcR and AtxA

Biochemical studies on Francisella tularensis RelA in (p)ppGpp biosynthesis

The bacterial stringent response is induced by nutrient deprivation and is mediated by enzymes of the RSH (RelA/SpoT homologue; RelA, (p)ppGpp synthetase I; SpoT, (p)ppGpp synthetase II) superfamily that control concentrations of the 'alarmones' (p)ppGpp (guanosine penta- or tetra-phosphate). This regulatory pathway is present in the vast majority of pathogens and has been proposed as a potential anti-bacterial target. Current understanding of RelA-mediated responses is based on biochemical studies using Escherichia coli as a model. In comparison, the Francisella tularensis RelA sequence contains a truncated regulatory C-terminal region and an unusual synthetase motif (EXSD). Biochemical analysis of F. tularensis RelA showed the similarities and differences of this enzyme compared with the model RelA from Escherichia coli. Purification of the enzyme yielded a stable dimer capable of reaching concentrations of 10 mg/ml. In contrast with other enzymes from the RelA/SpoT homologue superfamily, activity assays with F. tularensis RelA demonstrate a high degree of specificity for GTP as a pyrophosphate acceptor, with no measurable turnover for GDP. Steady state kinetic analysis of F. tularensis RelA gave saturation activity curves that best fitted a sigmoidal function. This kinetic profile can result from allosteric regulation and further measurements with potential allosteric regulators demonstrated activation by ppGpp (5',3'-dibisphosphate guanosine) with an EC50 of 60±1.9 ?M. Activation of F. tularensis RelA by stalled ribosomal complexes formed with ribosomes purified from E. coli MRE600 was observed, but interestingly, significantly weaker activation with ribosomes isolated from Francisella philomiragia