Nicole J. Moreland

A highly conserved transcriptional repressor controls a large regulon involved in lipid degradation in Mycobacterium smegmatis and Mycobacterium tuberculosis

The Mycobacterium tuberculosis TetR‐type regulator Rv3574 has been implicated in pathogenesis as it is induced in vivo, and genome‐wide essentiality studies show it is required for infection. As the gene is highly conserved in the mycobacteria, we deleted the Rv3574 orthologue in Mycobacterium smegmatis (MSMEG_6042) and used real‐time quantitative polymerase chain reaction and microarray analyses to show that it represses the transcription both of itself and of a large number of genes involved in lipid metabolism. We identified a conserved motif within its own promoter (TnnAACnnGTTnnA) and showed that it binds as a dimer to 29 bp probes containing the motif. We found 16 and 31 other instances of the motif in intergenic regions of M. tuberculosis and M. smegmatis respectively. Combining the results of the microarray studies with the motif analyses, we predict that Rv3574 directly controls the expression of 83 genes in M. smegmatis, and 74 in M. tuberculosis. Many of these genes are known to be induced by growth on cholesterol in rhodococci, and palmitate in M. tuberculosis. We conclude that this regulator, designated elsewhere as kstR, controls the expression of genes used for utilizing diverse lipids as energy sources, possibly imported through the mce4 system.

A Flexible And Economical Medium-Throughput Strategy for Protein Production And Crystallization

Large-scale structural genomics centres rely heavily on robotics to ensure that maximum throughput is achieved. However, the size and cost of these approaches is out of the reach of most academic structural biology efforts. A major challenge for such groups is to adapt current high-throughput schemes to a reasonable scale with the resources available. A flexible medium-throughput approach has been developed that is suitable for typical academic research groups. Following nested PCR, targets are routinely cloned into two Gateway expression vectors (pDEST15 for an N-terminal GST tag and pDEST17 for an N-terminal His tag). Expression of soluble recombinant protein in Escherichia coli is rapidly assessed in 96-well format. An eight-probe sonicator is utilized and a six-buffer lysis screen was incorporated to enhance solubility. Robotics is reserved for crystallization, since this is the key bottleneck for crystallography. Screening proteins with a 480-condition protocol using a Cartesian nanolitre-dispensing robot has increased crystallization success markedly, with an overall success rate (structures solved out of proteins screened) of 19%. The methods are robust and economical -- with the exception of the crystallization robot, investment in additional equipment has been minimal at 9000 US dollars. All protocols are designed for individuals so that graduate students and postdoctoral fellows gain expertise in every aspect of the structural pipeline, from cloning to crystallization.

Crystal structures of F420-dependent glucose-6-phosphate dehydrogenase FGD1 involved in the activation of the anti-tuberculosis drug candidate PA-824 reveal the basis of coenzyme and substrate binding.

The modified flavin coenzyme F420 is found in a restricted number of microorganisms. It is widely distributed in mycobacteria, however, where it is important in energy metabolism, and in Mycobacterium tuberculosis (Mtb) is implicated in redox processes related to non-replicating persistence. In Mtb, the F420-dependent glucose-6-phosphate dehydrogenase FGD1 provides reduced F420 for the in vivo activation of the nitroimidazopyran prodrug PA-824, currently being developed for anti-tuberculosis therapy against both replicating and persistent bacteria. The structure of M. tuberculosis FGD1 has been determined by x-ray crystallography both in its apo state and in complex with F420 and citrate at resolutions of 1.90 and 1.95Å, respectively. The structure reveals a highly specific F420 binding mode, which is shared with several other F420-dependent enzymes. Citrate occupies the substrate binding pocket adjacent to F420 and is shown to be a competitive inhibitor (IC50 43 μm). Modeling of the binding of the glucose 6-phosphate (G6P) substrate identifies a positively charged phosphate binding pocket and shows that G6P, like citrate, packs against the isoalloxazine moiety of F420 and helps promote a butterfly bend conformation that facilitates F420 reduction and catalysis.

Status of research and development of vaccines for Streptococcus pyogenes

Streptococcus pyogenes is an important global pathogen, causing considerable morbidity and mortality, especially in low and middle income countries where rheumatic heart disease and invasive infections are common. There is a number of promising vaccine candidates, most notably those based on the M protein, the key virulence factor for the bacterium. Vaccines against Streptococcus pyogenes are considered as impeded vaccines because of a number of crucial barriers to development. Considerable effort is needed by key players to bring current vaccine candidates through phase III clinical trials and there is a clear need to develop a roadmap for future development of current and new candidates.

Working towards a group A streptococcal vaccine: report of a collaborative Trans-Tasman workshop.

Group A Streptococcus (GAS) infections represent a major public health burden in both developing and developed countries. In Australia and New Zealand GAS associated diseases are serious problems in Indigenous populations and a major cause of health inequality. Political recognition of these inequalities is providing impetus for strategies that reduce GAS disease and the development of a GAS vaccine now has governmental support in both Australia and New Zealand. Accordingly, an expert workshop was convened in March 2013 to consider available data on GAS vaccines. M-protein based vaccines constructed from the hyper-variable N-terminal region (30-valent vaccine) or the conserved C-repeat domain (J8 vaccine) were reviewed together with vaccine candidates identified using multi high-throughput approaches. Performing a comprehensive assessment of regional GAS strain epidemiology, defining the immune correlates of protection, and the establishment of clinical trial sites were identified as critical activities for a Trans-Tasman vaccine development programme.

M-protein analysis of Streptococcus pyogenes isolates associated with acute rheumatic fever in New Zealand

ABSTRACT We applied an emm cluster typing system to group A Streptococcus strains in New Zealand, including those associated with acute rheumatic fever (ARF). We observed few so-called rheumatogenic emm types but found a high proportion of emm types previously associated with pyoderma, further suggesting a role for skin infection in ARF.

Structural Conservation, Variability, and Immunogenicity of the T6 Backbone Pilin of Serotype M6 Streptococcus pyogenes

ABSTRACT Group A streptococcus (GAS; Streptococcus pyogenes) is a Gram-positive human pathogen that causes a broad range of diseases ranging from acute pharyngitis to the poststreptococcal sequelae of acute rheumatic fever. GAS pili are highly diverse, long protein polymers that extend from the cell surface. They have multiple roles in infection and are promising candidates for vaccine development. This study describes the structure of the T6 backbone pilin (BP; Lancefield T-antigen) from the important M6 serotype. The structure reveals a modular arrangement of three tandem immunoglobulin-like domains, two with internal isopeptide bonds. The T6 pilin lysine, essential for polymerization, is located in a novel VAKS motif that is structurally homologous to the canonical YPKN pilin lysine in other three- and four-domain Gram-positive pilins. The T6 structure also highlights a conserved pilin core whose surface is decorated with highly variable loops and extensions. Comparison to other Gram-positive BPs shows that many of the largest variable extensions are found in conserved locations. Studies with sera from patients diagnosed with GAS-associated acute rheumatic fever showed that each of the three T6 domains, and the largest of the variable extensions (V8), are targeted by IgG during infection in vivo. Although the GAS BP show large variations in size and sequence, the modular nature of the pilus proteins revealed by the T6 structure may aid the future design of a pilus-based vaccine.

Making sense of a missense mutation: characterization of MutT2, a Nudix hydrolase from Mycobacterium tuberculosis, and the G58R mutant encoded in W-Beijing strains of M. tuberculosis.

Recent polymorphism analyses of Mycobacterium tuberculosis strains have identified missense mutations unique to the W-Beijing lineage in genes belonging to the Nudix hydrolase superfamily. This study investigates the structure and function of one of these Nudix hydrolases, MutT2, and examines the effect that the W-Beijing mutation (G58R) has on enzyme characteristics. MutT2 has a preference for cytidine triphosphates, and although the G58R mutation does not alter nucleotide specificity, it reduces the proteins affinity for divalent cations. The K(D) of free Mg(2+) is 79-fold higher for the G58R mutant (3.30 +/- 0.19 mM) compared with that for the wild-type (41.7 +/- 1.4 microM). Circular dichroism and nuclear magnetic resonance spectroscopy measurements show that while the mutation does not perturb the overall structure of the protein, protein stability is significantly compromised by the presence of the arginine with DeltaG (H(2)O), the free-energy of unfolding, being reduced by 2.48 kcal mol(-1) in the G58R mutant. Homology modeling of MutT2 shows that Gly-58 is in close proximity (10.8 A) to the Mg(2+) binding site formed by the highly conserved Nudix box residues and hydrogen bonds with Ala-54 in the preceding alpha-helix. This may explain the increased divalent cation requirement and decreased stability observed when an arginine is substituted for glycine at this position. A role for MutT2 in the regulation of cytidine-triphosphates available for nucleotide-dependent reactions is postulated, and the impact that the G58R mutation may have on these reactions is discussed.

Development of Group A streptococcal vaccines: an unmet global health need.

ABSTRACT Group A Streptococcus (GAS) infections are a significant global cause of morbidity and mortality. GAS diseases disproportionally affect those living in conditions characterized by poverty and social injustice, in both developing countries and in marginalized populations of industrialized nations. In Australia and New Zealand, GAS-associated Acute Rheumatic Fever (ARF) is a major cause of health inequality disproportionally affecting indigenous children. Recognition of these inequalities by the governments of Australia and New Zealand has resulted in the formation of a Trans-Tasman Coalition to Advance New Vaccines for group A Streptococcus (CANVAS). This review provides an update on the current status of GAS vaccine development, and describes global efforts by CANVAS and others to accelerate the development of GAS vaccines.

Survey of the bp/tee genes from clinical group A streptococcus isolates in New Zealand – implications for vaccine development

Group A streptococcus (GAS) is responsible for a wide range of diseases ranging from superficial infections, such as pharyngitis and impetigo, to life-threatening diseases, such as toxic shock syndrome and acute rheumatic fever (ARF). GAS pili are hair-like extensions protruding from the cell surface and consist of highly immunogenic structural proteins: the backbone pilin (BP) and one or two accessory pilins (AP1 and AP2). The protease-resistant BP builds the pilus shaft and has been recognized as the T-antigen, which forms the basis of a major serological typing scheme that is often used as a supplement to M typing. A previous sequence analysis of the bp gene (tee gene) in 39 GAS isolates revealed 15 different bp/tee types. In this study, we sequenced the bp/tee gene from 100 GAS isolates obtained from patients with pharyngitis, ARF or invasive disease in New Zealand. We found 20 new bp/tee alleles and four new bp/tee types/subtypes. No association between bp/tee type and clinical outcome was observed. We confirmed earlier reports that the emm type and tee type are associated strongly, but we also found exceptions, where multiple tee types could be found in certain M/emm type strains, such as M/emm89. We also reported, for the first time, the existence of a chimeric bp/tee allele, which was assigned into a new subclade (bp/tee3.1). A strong sequence conservation of the bp/tee gene was observed within the individual bp/tee types/subtypes (>97 % sequence identity), as well as between historical and contemporary New Zealand and international GAS strains. This temporal and geographical sequence stability provided further evidence for the potential use of the BP/T-antigen as a vaccine target.