Investigating the Effects of N-Methyl Modifications on Activity of a Truncated Group I Competence Stimulating Peptide (CSP1) on Quorum Sensing in Streptococcus pneumoniae

Bacterial infections are becoming increasingly difficult to treat as more bacteria develop antibiotic resistance. Our research aims to produce a therapeutic to block bacterial communication, rendering bacteria non-pathogenic without killing them, thus avoiding driving the evolution of resistant strains. Bacteria communicate through a phenomenon called quorum sensing, in which bacteria release signal molecules to indicate their population size and density. Once a population is large enough, it engages in behaviors that are effective only when the whole group, rather than individual bacterium, exhibit them. This phenomenon can induce previously non-pathogenic bacteria populations to attack their hosts. S. pneumoniae uses a 17-amino acid long peptide called competence stimulating peptide (CSP) to communicate. At a threshold concentration, CSP binds and activates a receptor called comD, starting a signaling cascade ending with bacteria exhibiting group behaviors such as virulence. CSP analogs that outcompete the native peptide for binding to comD could impede bacterial communication, and therefore, pathogenicity. However, finding an effective therapeutic is complicated by the fact that different strains of S. pneumoniae have different signaling molecules called CSP-1 and CSP-2 that will only bind respectively to comD-1 and comD-2, respectively. Our previous research has shown CSP-1 interacts slightly more effectively with comD-2 than CSP-2 will with comD-1, and that the final two residues on both peptides are unnecessary for binding. Therefore, the purpose of this project was to complete an N-methyl scan of a 15-amino acid long CSP-1 analog to determine the importance of different backbone hydrogen bonds on the activity of the peptide. Solid-phase peptide synthesis was utilized to construct a library of 15 N-methyl analogs, and cell-based reporter assays were conducted to evaluate the ability of the different analogs to modulate quorum sensing in both S. pneumoniae specificity groups

Rubisco function, evolution, and engineering

Carbon fixation is the process by which CO2 is converted from a gas into biomass. The Calvin Benson Bassham (CBB) cycle is the dominant carbon fixation pathway on earth, driving >99.5% of the ~120 billion tons of carbon that are "fixed" as sugar, by plants, algae and cyanobacteria. The carboxylase enzyme in the CBB, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fixes one CO2 molecule per turn of the cycle. Despite being critical to the assimilation of carbon, rubisco's kinetic rate is not very fast and it is a bottleneck in flux through the pathway. This presents a paradox - why hasn't rubisco evolved to be a better catalyst? Many hypothesize that the catalytic mechanism of rubisco is subject to one or more trade-offs, and that rubisco variants have been optimized for their native physiological environment. Here we review the evolution and biochemistry of rubisco through the lens of structure and mechanism in order to understand what trade-offs limit its improvement. We also review the many attempts to improve rubisco itself and, thereby, promote plant growth

Rubisco Function, Evolution, and Engineering.

Carbon fixation is the process by which CO2 is converted from a gas into biomass. The Calvin-Benson-Bassham cycle (CBB) is the dominant carbon-consuming pathway on Earth, driving >99.5% of the ∼120 billion tons of carbon that are converted to sugar by plants, algae, and cyanobacteria. The carboxylase enzyme in the CBB, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fixes one CO2 molecule per turn of the cycle into bioavailable sugars. Despite being critical to the assimilation of carbon, rubisco's kinetic rate is not very fast, limiting flux through the pathway. This bottleneck presents a paradox: Why has rubisco not evolved to be a better catalyst? Many hypothesize that the catalytic mechanism of rubisco is subject to one or more trade-offs and that rubisco variants have been optimized for their native physiological environment. Here, we review the evolution and biochemistry of rubisco through the lens of structure and mechanism in order to understand what trade-offs limit its improvement. We also review the many attempts to improve rubisco itself and thereby promote plant growth

Structure–Activity Relationships of the Competence Stimulating Peptides (CSPs) in <i>Streptococcus pneumoniae</i> Reveal Motifs Critical for Intra-group and Cross-group ComD Receptor Activation

<i>Streptococcus pneumoniae</i> is a highly recombinogenic human pathogen that utilizes the competence stimulating peptide (CSP)-based quorum sensing (QS) circuitry to acquire antibiotic resistance genes from the environment and initiate its attack on the human host. Modulation of QS in this bacterium, either inhibition or activation, can therefore be used to attenuate <i>S. pneumoniae</i> infectivity and slow down pneumococcal resistance development. In this study, we set to determine the molecular mechanism that drives CSP:receptor binding and identify CSP-based QS modulators with distinct activity profiles. To this end, we conducted systematic replacement of the amino acid residues in the two major CSP signals (CSP1 and CSP2) and assessed the ability of the mutated analogs to modulate QS against both cognate and noncognate ComD receptors. We then evaluated the overall 3D structures of these analogs using circular dichroism (CD) to correlate between the structure and function of these peptides. Our CD analysis revealed a strong correlation between α-helicity and bioactivity for both specificity groups (CSP1 and CSP2). Furthermore, we identified the first pan-group QS activator and the most potent group-II QS inhibitor to date. These chemical probes can be used to study the role of QS in <i>S. pneumoniae</i> and as scaffolds for the design of QS-based anti-infective therapeutics against <i>S. pneumoniae</i> infections

Discovery and characterization of a novel family of prokaryotic nanocompartments involved in sulfur metabolism.

Prokaryotic nanocompartments, also known as encapsulins, are a recently discovered proteinaceous organelle-like compartment in prokaryotes that compartmentalize cargo enzymes. While initial studies have begun to elucidate the structure and physiological roles of encapsulins, bioinformatic evidence suggests that a great diversity of encapsulin nanocompartments remains unexplored. Here, we describe a novel encapsulin in the freshwater cyanobacterium Synechococcus elongatus PCC 7942. This nanocompartment is upregulated upon sulfate starvation and encapsulates a cysteine desulfurase enzyme via an N-terminal targeting sequence. Using cryo-electron microscopy, we have determined the structure of the nanocompartment complex to 2.2 Å resolution. Lastly, biochemical characterization of the complex demonstrated that the activity of the cysteine desulfurase is enhanced upon encapsulation. Taken together, our discovery, structural analysis, and enzymatic characterization of this prokaryotic nanocompartment provide a foundation for future studies seeking to understand the physiological role of this encapsulin in various bacteria

Discovery and characterization of a novel family of prokaryotic nanocompartments involved in sulfur metabolism.

Prokaryotic nanocompartments, also known as encapsulins, are a recently discovered proteinaceous organelle-like compartment in prokaryotes that compartmentalize cargo enzymes. While initial studies have begun to elucidate the structure and physiological roles of encapsulins, bioinformatic evidence suggests that a great diversity of encapsulin nanocompartments remains unexplored. Here, we describe a novel encapsulin in the freshwater cyanobacterium Synechococcus elongatus PCC 7942. This nanocompartment is upregulated upon sulfate starvation and encapsulates a cysteine desulfurase enzyme via an N-terminal targeting sequence. Using cryo-electron microscopy, we have determined the structure of the nanocompartment complex to 2.2 Å resolution. Lastly, biochemical characterization of the complex demonstrated that the activity of the cysteine desulfurase is enhanced upon encapsulation. Taken together, our discovery, structural analysis, and enzymatic characterization of this prokaryotic nanocompartment provide a foundation for future studies seeking to understand the physiological role of this encapsulin in various bacteria