STEM 1 is probably the most unique science course I have ever taken. Taught and guided by Dr. Crowthers, in this class, every student develops their own independent research project to examine a topic of their interest in depth. Through this course, I have learned how to better brainstorm, research, and communicate my project ideas. We start with simple ideas, and throughout the course of 6 month, we develop our initial idea into a full project, complete with thorough research into our field of interest. We prepare for presentations at our December Fair, which is a preliminary fair to provide an update on our project and practice presenting our work. Through winter break and January, we finish our data collection and analysis in order to prepare for our February Fair, where we get to present our complete project. Take a look below to learn about my project!
Classifying the functionality of differentially expressed genes as a mechanism of resistance in Shigella flexneri to bile.
Researcher: Kiara Lavana
Advisors: Dr. Christina Faherty, Ph.D. (Massachusetts General Hospital), Dr. Kevin Crowthers, Ph.D. (Massachusetts Academy of Math and Science)
My project focuses on delineating the pathogenesis of Shigella flexneri, an enteric pathogen. I partnered with the Faherty Lab at the Massachusetts General Hospital to study the transcriptional alterations occurring in S. flexneri post-bile exposure. I utilized a mathematical model to isolate different gene clusters in order to predict the functions of proteins within different groupings.
Shigella flexneri is a Gram-negative, facultative intracellular pathogen targeting the colonic epithelium within the human gastrointestinal tract. Shigellosis, the infection associated with Shigella’s proliferation, is a major cause of diarrhea and bacterial dysentery. During its gastrointestinal transit, S. flexneri employs mechanisms that enable its survival in anaerobic environments and evasion of bile, a digestive fluid produced to reduce pathogenic bacterial concentrations. This study aimed to delineate the functions of differentially expressed S. flexneri genes that were altered in the presence of bile. It was hypothesized that differentially expressed gene clusters would provide crucial information on understanding Shigella’s ability to evade biological control mechanisms. Through using variable input gamma parameters in a mathematical model, differentially expressed genes were identified and further "hypothetical" or undefined proteins were evaluated. BLAST sequence analysis and database research was performed to hypothesize the function of these undefined proteins. 61 repressed genes were isolated from the bile-exposed S. flexneri cultures. There was significant down-regulation of gene operons associated with maintaining acid tolerance (most presumably employed in the acidic environments of the stomach). Genes associated with maintaining homeostasis in low pH environments, such as ydeP, gadC and gadE, along with transcriptional regulators such as ydeO, were postulated to have been repressed due to Shigella’s transition to the neutral, almost alkaline environment of the colon. This finding reveals an integral facet of S. flexneri’s transit, which includes its ability to repress unnecessary genes to conserve energy for the production of necessary proteins. The functionality of the proteins enabling Shigella pathogenesis will support future drug development by characterizing potential pharmaceutical targets for drug inhibitors. This helps combat not only shigellosis, but also antibiotic resistance since the resistance from environmental stimuli to therapeutics is often conferred in the bacteria.
What are the functions of differentially expressed Shigella flexneri genes in the presence of bile in the human gastrointestinal tract?
If a mathematical model is utilized to distinguish differentially expressed gene clusters in S. flexneri after exposure to bile, then the function of the collective gene cluster will provide insight to the functionality of the hypothetical protein.
During the host gastrointestinal transit, one of the numerous stimuli S. flexneri comes into contact with is bile, a digestive fluid composed of various proteins, lipids, carbohydrates, mineral salts, and vitamins. Of these components, bile salts, or conjugated bile acids to glycine or taurine through N-acyl amidation, are known to provide protection against pathogenic bacteria (Merritt & Donaldson, 2009). Concentrations of bile salts in the small intestine range from 0.2% to 2% (wt/vol) depending on time of day, diet, and other individual variances. Bile is bactericidal and plays a distinct role in preventing the proliferation of pathogenic bacterial strains. Despite this, Gram-negative bacteria, have adapted stress responses that enable their survival and continued proliferation in the stressful environment with high concentrations of bile; these enteropathogens have even begun utilizing host components such as bile as localization signals to regulate virulence gene expression and enhance their infection capacity while in transit (Nickerson et al., 2017).
Shigella flexneri is a species of Gram-negative, facultative intracellular pathogens that target the colonic epithelium within the human gastrointestinal tract (Köseoğlu, et al, 2019). Shigellosis, the infection that occurs with the proliferation of S. flexneri in the intestines, causes significant morbidity and mortality around the world each year, predominantly in children under 5 years old in developing countries lacking access to proper sanitation and treatment facilities. Thus far, no vaccine against shigellosis exists to effectively treat the infection, and with the emergence of antibiotic resistance, more complications arise when trying to reduce bacterial numbers.
While many aspects of the Shigella invasion process, intracellular survival, and immune response have been studied thoroughly, less literature explicates the mechanisms behind the bacterium transit to the colonic epithelium in order to establish infection. It is known that S. flexneri utilize a type III secretion system and lpa proteins, and other effector proteins to establish and then subsequently maintain infection (Nickerson et al., 2017).
Gram-negative bacteria, including the Shigella strains, utilize distinct stress responses to resist the bactericidal effects of high bile salt concentrations. These mechanisms include the expulsion of bile compounds with efflux machinery, biofilm formation, activation of stress-response genes, and activation of DNA repair systems through different gene networks. Differential gene expression accounts for the repression and induction of various genes and proteins postulated to help the bacteria survive in a harsh environment. However, despite these changes in gene expression, still many “hypothetical” or undefined proteins exist; classifying the function of these proteins in relation to aid the bacteria survive in harsh conditions is crucial to delineating the mechanisms of resistance employed by S. flexneri.
A mathematical algorithm was implemented in a computer program to take in input gene data and distinguish differentially expressed genes (defined as induced or repressed genes with a 2-fold or greater change in expression and a P-value cutoff of ≤ 0.05). After initializing the program to isolate only repressed genes, the ideal gamma parameter was determined through trial and error. A higher gamma will output more gene clusters, but the genes in clusters will be more closely related. Conversely, decreasing the gamma creates broader gene clusters, but the associations between the proteins may not be as closely correlated. A gamma parameter of 2.1 was elected to be used since it yielded similar results +/- 1 degrees.
With the list of the repressed gene clustered into different groups, the next stage in the process was performing database research in order to hypothesize a function for the gene cluster and any hypothetical proteins. Using the KEGG Pathway database, the orthologs of specific genes BLAST sequences analysis against other S. flexneri and Enterobactericcae strains was used to generalize the function of each gene and how they aid Shigella transit. The annotations from different databases provided previous papers and information on the genes. These studies were referenced to provide reasoning for a gene’s repression.
Dynamic representation of transcriptionally altered gene clusters. With a specified gamma parameter of 2.1, repressed gene clusters with a fold change ≤ -2.00 and P-value of ≤0.05 were clustered from a set of Shigella flexneri genes post-exposure to bile. 12 gene clusters are plotted, along with the computed connections between each gene. Cluster 1, Cluster 2, Cluster 5, Cluster 6, and Cluster 7 were isolated as genes of interest due to their high number of connections to other genes.
Summary table of all predictive functions of genes of interest. Out of the 29 genes of interest, these 11 genes exhibited unique relationships with other genes and cellular functions. The full list of gene predicted roles can be found in my STEM Thesis writeup.
Glutamate-dependent acid resistance in S. flexneri. Genes S2649 and S2648 are a decarboxylase and antiporter involved with exchanging extracellular glutamate for intracellular gamma-aminobutyric acid (GABA) under acidic conditions, which requires H+ to be uptaken as a reactant, thereby raising the pH inside S. flexneri and protecting the cell from low pH environments. Various genes in Cluster 1 were associated with glutamate-dependent acid resistance.
A majority of the hya operon was downregulated subsequent to bile salt exposure in S. flexneri. Expression of hya is increased under anaerobic fermentative conditions and decreased under aerobic or anaerobic respiratory conditions with nitrate.
The genes of interest out of the full 61 repressed genes chosen for further analysis in this study. The genes in Clusters 1, 2, 7 were chosen because of their unique relationships and associations with other clusters and genes. For example, Cluster 7 appeared to cluster at an intersection of multiple other clusters.
From the predicted roles of downregulated genes, a few major continuities stood out between some clusters.
From the amount of acid tolerance and actid-dependent proteins (11 genes), it can be reasonably inferred that Cluster 1 groups proteins associated with acid-resistance. While enteric bacteria like S. flexneri adhere and proliferate within the colonic epithelium, their colonization necessitates the transient survival within the stomach when in transit through the gastrointestinal tract. Therefore, when entering the neutral, almost alkaline environment of the intestines (moderate range from pH 5-8), it remains likely that S. flexneri would down-regulate unnecessary protective responses designed to aid bacteria survival in low-pH environments.
Various genes characteristic to stationary phase survival were downregulated and clustered in Cluster 2. For example, a hypothetical protein (S4448) predicted to function as an anti-adapter protein IraD mediating stability after DNA damage has historically been upregulated during the transition to stationary phase in enteric bacteria according to Merrikh et al. IraD promotes the post-translation stability of RpoS through the inhibition of adapter protein RssB. Similarly, the chaperone-modulator protein CbpM (S1072) works with CbpA to regulate the activity of the DnaK chaperone system after maximal transcription during the transition from exponential growth to stationary phase. Further, the global regulator DNA starvation/stationary phase protection protein Dps (S0805) also protects DNA from oxidative stress during the stationary phase, suggesting the Cluster 2 may group genes together that function for stationary phase survival. Bacteria like S. flexneri have developed a variety of mechanisms to survive in nutrient-depleted and harsh environments like the stomach. As a consequence of prolonged starvation, bacterial species may also enter a dynamic nonproliferative state known as a stationary phase where transcriptional alterations occur to prime for a more resistance cell.
Multiple genes from the hydrogenase 1 (hya operon) were repressed subsequent to bile exposure, such as hydrogenase 1 large subunit (S1041), hydrogenase-1 small subunit (S1040), hydrogenase-1 operon protein HyaE (S1044). These proteins have roles in hydrogen cycling during fermentative growth, a necessary product within the stomach since low pH environments usually possess a high concentration of fermentative acids. Expression of hya is increased under anaerobic fermentative conditions and decreased under aerobic or anaerobic respiratory conditions with nitrate.
These findings have the potential to delineate the mechanisms by which Shigella and other enteropathogens survive through their transit long enough to establish an infection. In comparison to other enteropathogens, the Shigella strain is highly understudied; limited literature exists on Shigella mechanisms of infection and resistance to natural stimuli in the body and their remains a significant gap in knowledge regarding how the bacterium successfully reaches the colonic epithelium to establish infection. The primary findings of this study point to an important facet in Shigella survival; their downregulation in the small intestines of genes designed to aid the bacterium for low-pH survival highlight the transition from the stomach to the small intestines as a potential future area of research. By systematically addressing the function of repressed Shigella genes through an extensive literature review, this study addresses the novel characterization of Shigella flexneri genes after bile salt exposure.
Further, this research could also lend itself to delineating the mechanism of antibiotic resistance in Shigella flexneri. The increased incidence of antibiotic resistance in enteric diseases complicates bacterial infection control, perpetuating longer hospitalization and higher healthcare costs for patients afflicted by these formerly treatable diseases. Oftentimes, bacterial resistance to environmental regulators such as bile salts confers multidrug resistance, which then enables longer and more extensive infection periods; this cyclic process threatens health safety globally, making a greater understanding of bacterial mechanisms of resistance crucial for the development of improved therapeutics and more effective antimicrobial agents.
Future analysis is warranted to validate the hypotheses generated in this study. A significant portion of the findings postulated integral transcriptional alterations occurring during the transition from the low-pH environments of the stomach to the neutral, almost alkaline environments of the intestinal tract. Specific phenotypic assays designed to simulate the bacterial growth conditions of the stomach is warranted to substantiate the hypotheses generated in this experiment. Further study of this transition can hopefully provide more insight into potential therapy targets for shigellosis in the future. Ultimately, this study provided the foundation for a long term project analyzing the transcriptional alterations occurring in Shigella by producing potential hypotheses and reasonings for the gene’s downregulation.
This study aimed to isolate clusters of downregulated genes in Shigella flexneri after bile salt exposure and utilize their shared pathways and similarity to other genes to hypothesize their function in Shigella survival during intestinal transit. A mathematical model used RNA-seq analysis data to first distinguish differentially expressed genes (defined as down-regulated or upregulated with a fold change of ≥ 2.0 or ≥ - 2.0 and a P value cutoff of ≤0.05), and then utilize an algorithm to group the genes into different clusters based on the strength of their associations. A full list of the repressed genes can be found in Appendix A. Once specific genes were identified, BLAST sequence tools on the NIH and UniProt databases were used to find homologous proteins in other Shigella or other enteropathogenic strains. Database annotations from the Kyoto Encyclopedia of Genes and Genomes were used to locate relevant studies explaining the function of homologous proteins in other bacterial strains. Major findings and results from papers were used to formulate general hypotheses of different gene functions and a potential reasoning for their downregulation.
12 distinct gene clusters were distinguished by the mathematical model. Clusters 1, 2, and 7 exhibited relatively defined functionalities based on the genes in their clusters. Cluster 1 was postulated to be involved with acid-tolerance in the bacteria, with an emphasis on glutamate-dependent acid-resistance in many of the proteins. Cluster 2 possessed multiple genes associated with aiding the cell during a transition from exponential growth to stationary phase, which is relevant when considering the growth patterns of Shigella flexneri in the stomach and intestinal tract. Finally, Cluster 1, 2, 7 together possessed almost the entirety of the hya operon, a complex associated with hydrogen cycle and energy production during fermentative growth.
The novel characterization of Shigella flexneri’s genome will eventually aid treatment development for shigellosis and other enteropathogenic diseases. The transcription alteration exhibited by Shigella through their transit provides integral insight into the mechanisms by which bacteria evade the body’s natural defense system to establish an infection. These mechanisms can emerge as potential drug targets for future therapies. Further, the significance of the transition from the acidic stomach to neutral intestines could serve as a research area for future studies. Finally, understanding the mechanisms exhibited by Shigella to resist bile salts will only aid understanding of the multidrug resistance mechanisms also exhibited during treatment. Often, exposure to natural stimuli like bile salts can induce transcriptional alterations to the cell that effectively prime the bacterium against future antimicrobial agents.