Enzymatic Activities and DNA Substrate Specificity of Mycobacterium tuberculosis DNA Helicase XPB

XPB, also known as ERCC3 and RAD25, is a 3′→5′ DNA repair helicase belonging to the superfamily 2 of helicases. XPB is an essential core subunit of the eukaryotic basal transcription factor complex TFIIH. It has two well-established functions: in the context of damaged DNA, XPB facilitates nucleotide excision repair by unwinding double stranded DNA (dsDNA) surrounding a DNA lesion; while in the context of actively transcribing genes, XPB facilitates initiation of RNA polymerase II transcription at gene promoters. Human and other eukaryotic XPB homologs are relatively well characterized compared to conserved homologs found in mycobacteria and archaea. However, more insight into the function of bacterial helicases is central to understanding the mechanism of DNA metabolism and pathogenesis in general. Here, we characterized Mycobacterium tuberculosis XPB (Mtb XPB), a 3′→5′ DNA helicase with DNA-dependent ATPase activity. Mtb XPB efficiently catalyzed DNA unwinding in the presence of significant excess of enzyme. The unwinding activity was fueled by ATP or dATP in the presence of Mg2+/Mn2+. Consistent with the 3′→5′ polarity of this bacterial XPB helicase, the enzyme required a DNA substrate with a 3′ overhang of 15 nucleotides or more. Although Mtb XPB efficiently unwound DNA model substrates with a 3′ DNA tail, it was not active on substrates containing a 3′ RNA tail. We also found that Mtb XPB efficiently catalyzed ATP-independent annealing of complementary DNA strands. These observations significantly enhance our understanding of the biological roles of Mtb XPB

Biochemical Characterization of the DNA Helicases RecG, XPB and DinG in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis, a deadly human disease that kills millions of people every year. Mtb is notable for its survival and proliferation inside the harsh environment of the human macrophage, where many bacterial pathogens normally perish. This pathogen has an efficient DNA repair system that helps to maintain the integrity of its genome despite the severe genotoxic stress inside the macrophage, thereby enabling its intracellular survival and proliferation. Helicases, motor enzymes that unwind duplex nucleic acid strands into single strands, are vital enzymes required in virtually every aspect of nucleic acid metabolism, including DNA replication, repair, recombination and transcription. In this thesis, biochemical characterization of three Mycobacterium tuberculosis helicases; namely RecG, XPB and DinG is reported. This study specifically focussed on examining the DNA substrate specificity, ATPase activity, strand annealing activity, and nucleoside triphosphate and divalent metal preferences of these helicases. A number of site-specific mutations in various motifs of RecG was also constructed and their effects assessed. The study also examined the relative expression level of the genes encoding RecG, XPB and DinG helicases under a variety of genotoxic stress conditions. The studies presented in this thesis provide important insights into the function and possible roles of these helicases in M. tuberculosis and facilitate subsequent attempts to delineate the precise roles of RecG, XPB and DinG in Mtb genome maintenance and cellular fitness

Vaccine Candidate Double Mutant Variants of Enterotoxigenic Escherichia coli Heat-Stable Toxin

Heat-stable enterotoxin (ST) producing enterotoxigenic Escherichia coli (ETEC) strains are among the top four enteropathogens associated with moderate-to-severe diarrhea in children under five years in low-to-middle income countries, thus making ST a target for an ETEC vaccine. However, ST must be mutated to abolish its enterotoxicity and to prevent a potential immunological cross-reaction due to its structural resemblance to the human peptides uroguanylin and guanylin. To reduce the risk of eliciting cross-reacting antibodies with our lead STh-A14T toxoid, L9 was chosen as an additional mutational target. A double mutant vaccine candidate immunogen, STh-L9A/A14T, was constructed by conjugation to the synthetic virus-like mi3 nanoparticle using the SpyTag/SpyCatcher technology. This immunogen elicited STh neutralizing antibodies in mice, but with less consistency than STh-A14T peptide control immunogens. Moreover, individual sera from mice immunized with both single and double mutant variants displayed varying levels of unwanted cross-reacting antibodies. The lowest levels of cross-reacting antibodies were observed with STh-L9K/A14T control immunogens, suggesting that it is indeed possible to reduce the risk of eliciting cross-reacting antibodies by mutation. However, mutant-specific antibodies were observed for most double mutant immunogens, demonstrating the delicate balancing act between disrupting cross-reacting epitopes, keeping protective ones, and avoiding the formation of neoepitopes

Vaccine Candidate Double Mutant Variants of Enterotoxigenic Escherichia coli Heat-Stable Toxin

Heat-stable enterotoxin (ST) producing enterotoxigenic Escherichia coli (ETEC) strains are among the top four enteropathogens associated with moderate-to-severe diarrhea in children under five years in low-to-middle income countries, thus making ST a target for an ETEC vaccine. However, ST must be mutated to abolish its enterotoxicity and to prevent a potential immunological cross-reaction due to its structural resemblance to the human peptides uroguanylin and guanylin. To reduce the risk of eliciting cross-reacting antibodies with our lead STh-A14T toxoid, L9 was chosen as an additional mutational target. A double mutant vaccine candidate immunogen, STh-L9A/A14T, was constructed by conjugation to the synthetic virus-like mi3 nanoparticle using the SpyTag/SpyCatcher technology. This immunogen elicited STh neutralizing antibodies in mice, but with less consistency than STh-A14T peptide control immunogens. Moreover, individual sera from mice immunized with both single and double mutant variants displayed varying levels of unwanted cross-reacting antibodies. The lowest levels of cross-reacting antibodies were observed with STh-L9K/A14T control immunogens, suggesting that it is indeed possible to reduce the risk of eliciting cross-reacting antibodies by mutation. However, mutant-specific antibodies were observed for most double mutant immunogens, demonstrating the delicate balancing act between disrupting cross-reacting epitopes, keeping protective ones, and avoiding the formation of neoepitopes.publishedVersio

Enigmatic Pilus-Like Endospore Appendages of Bacillus cereus Group Species

The endospores (spores) of many Bacillus cereus sensu lato species are decorated with multiple hair/pilus-like appendages. Although they have been observed for more than 50 years, all efforts to characterize these fibers in detail have failed until now, largely due to their extraordinary resilience to proteolytic digestion and chemical solubilization. A recent structural analysis of B. cereus endospore appendages (Enas) using cryo-electron microscopy has revealed the structure of two distinct fiber morphologies: the longer and more abundant “Staggered-type” (S-Ena) and the shorter “Ladder-like” type (L-Ena), which further enabled the identification of the genes encoding the S-Ena. Ena homologs are widely and uniquely distributed among B. cereus sensu lato species, suggesting that appendages play important functional roles in these species. The discovery of ena genes is expected to facilitate functional studies involving Ena-depleted mutant spores to explore the role of Enas in the interaction between spores and their environment. Given the importance of B. cereus spores for the food industry and in medicine, there is a need for a better understanding of their biological functions and physicochemical properties. In this review, we discuss the current understanding of the Ena structure and the potential roles these remarkable fibers may play in the adhesion of spores to biotic and abiotic surfaces, aggregation, and biofilm formation

Development of an enterotoxigenic Escherichia colivaccine based on the heat-stable toxin

Infection with enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea-related illness and death among children under 5 years of age in low– and middle-income countries (LMIC). Recent studies have found that it is the ETEC subtypes that produce the heat-stable enterotoxin (ST), irrespective of whether they also secrete the heat-labile enterotoxin (LT), which contribute most importantly to the disease burden in children from LMIC. Therefore, adding an ST toxoid would importantly complement ongoing ETEC vaccine development efforts. The ST’s potent toxicity, its structural similarity to the endogenous peptides guanylin and uroguanylin, and its poor immunogenicity have all complicated the advancement of ST-based vaccine development. Recent remarkable progress, however, including the unprecedented screening for optimal ST mutants, mapping of cross-reacting ST epitopes and improved ST-carrier coupling strategies (bioconjugation and genetic fusion), enables the rational design of safe, immunogenic, and well-defined ST-based vaccine candidates

Purification and Characterization of Native and Vaccine Candidate Mutant Enterotoxigenic Escherichia coli Heat-Stable Toxins

Enterotoxigenic Escherichia coli (ETEC), which secretes the heat-stable toxin (ST) is among the four most important enteropathogens that cause moderate-to-severe diarrhea in children in low- and middle-income countries. ST is an intestinal molecular antagonist causing diarrhea and hence an attractive vaccine target. A non-toxic and safe ST vaccine should include one or more detoxifying mutations, and rigorous characterization of such mutants requires structurally intact peptides. To this end, we established a system for purification of ST and ST mutants by fusing the sequence encoding the mature ST peptide to the disulfide isomerase DsbC. A Tobacco Etch Virus protease cleavage site facilitates the proteolytic release of free ST with no additional residues. The purified ST peptides have the expected molecular masses, the correct number of disulfide bridges, and have biological activities and antigenic properties comparable to ST isolated from ETEC. We also show that free DsbC can assist in refolding denatured and misfolded ST in vitro. Finally, we demonstrate that the purification system can be used to produce ST mutants with an intact neutralizing epitope, that two single mutations, L9S and A14T, reduce toxicity more than 100-fold, and that the L9S/A14T double mutant has no measurable residual toxicity

Endospore Appendages: a novel pilus superfamily from the endospores of pathogenic Bacilli

Bacillus cereus sensu lato is a group of Gram-positive endospore-forming bacteria with high ecological diversity. Their endospores are decorated with micrometer-long appendages of unknown identity and function. Here, we isolate endospore appendages (Enas) from the food poisoning outbreak strain B. cereus NVH 0075-95 and find proteinaceous fibers of two main morphologies: S- and L-Ena. By using cryoEM and 3D helical reconstruction of S-Enas, we show these to represent a novel class of Gram-positive pili. S-Enas consist of single domain subunits with jellyroll topology that are laterally stacked by β-sheet augmentation. S-Enas are longitudinally stabilized by disulfide bonding through N-terminal connector peptides that bridge the helical turns. Together, this results in flexible pili that are highly resistant to heat, drought, and chemical damage. Phylogenomic analysis reveals a ubiquitous presence of the ena-gene cluster in the B. cereus group, which include species of clinical, environmental, and food importance. We propose Enas to represent a new class of pili specifically adapted to the harsh conditions encountered by bacterial spores.publishedVersio

Lineage-specific proteomic signatures in the Mycobacterium tuberculosis complex reveal differential abundance of proteins involved in virulence, DNA repair, CRISPR-Cas, bioenergetics and lipid metabolism

Despite the discovery of the tubercle bacillus more than 130 years ago, its physiology and the mechanisms of virulence are still not fully understood. A comprehensive analysis of the proteomes of members of the human-adapted Mycobacterium tuberculosis complex (MTBC) lineages 3, 4, 5, and 7 was conducted to better understand the evolution of virulence and other physiological characteristics. Unique and shared proteomic signatures in these modern, pre-modern and ancient MTBC lineages, as deduced from quantitative bioinformatics analyses of high-resolution mass spectrometry data, were delineated. The main proteomic findings were verified by using immunoblotting. In addition, analysis of multiple genome alignment of members of the same lineages was performed. Label-free peptide quantification of whole cells from MTBC lineages 3, 4, 5, and 7 yielded a total of 38,346 unique peptides derived from 3092 proteins, representing 77% coverage of the predicted proteome. MTBC lineage-specific differential expression was observed for 539 proteins. Lineage 7 exhibited a markedly reduced abundance of proteins involved in DNA repair, type VII ESX-3 and ESX-1 secretion systems, lipid metabolism and inorganic phosphate uptake, and an increased abundance of proteins involved in alternative pathways of the TCA cycle and the CRISPR-Cas system as compared to the other lineages. Lineages 3 and 4 exhibited a higher abundance of proteins involved in virulence, DNA repair, drug resistance and other metabolic pathways. The high throughput analysis of the MTBC proteome by super-resolution mass spectrometry provided an insight into the differential expression of proteins between MTBC lineages 3, 4, 5, and 7 that may explain the slow growth and reduced virulence, metabolic flexibility, and the ability to survive under adverse growth conditions of lineage 7.publishedVersio