Undergraduate Journal of Experimental Microbiology and Immunology

Staphylococcus epidermidis biofilm growth in response to glucose: Implications for diabetic implantable device infections

Jenna Buragina — 2023-09-05

The association between high bacterial infection rates and diabetes mellitus (DM) has been well established, where DM specifically predisposes individuals to frequent and severe biofilm-associated implantable device infections (IDI). Diabetic hyperglycemia, or elevated blood sugar, is accepted to contribute to this phenomenon and thus has prompted questions regarding how biofilm development is enhanced in a glucose-rich environment. Staphylococcus epidermidis is the species responsible for most IDI, however, few studies have evaluated the effect of excess glucose on the biofilm formation of this bacterium and have not considered how low oxygen and pH, the physiologically relevant characteristics of IDI, may affect results. Here, S. epidermidis was grown in media containing 0, 20, 200, and 2000 mg/dL of glucose under aerobic and anaerobic conditions. Biofilm mass was quantified using the microtiter crystal violet assay and media pH was measured prior to and following biofilm growth. Glucose significantly increased biofilm mass only in anaerobic conditions at 2000 mg/dL, where increasing glucose concentrations corresponded with progressive media acidification. These data identify a relationship between oxygen condition, reduced pH, and enhanced S. epidermidis biofilm mass under excess glucose conditions, and thus provide insight into different variables that may contribute to the link between IDI and DM.

Recombinant Chitinase C produced through cell-free protein synthesis retains chitin-binding capabilities

Jen Wang — 2023-09-05

Optimizing high-yield production of target proteins is often a time-consuming process for many experiments. Cell-free protein synthesis (CFPS) is a potential solution, as it eliminates preliminary processes, such as cell transformation, while providing high protein concentration. In this study, our target protein for CFPS production was Chitinase C (ChiC) from Pseudomonas aeruginosa PAO1. We hypothesized that ChiC would retain its chitin-binding functionality through CFPS synthesis. Our study demonstrated that CFPS can successfully produce recombinant ChiC in Escherichia coli BL21 (DE3) cell extracts that not only fold at the appropriate size, but also possess chitin-binding functionality. Overall, CFPS was revealed as a viable avenue for ChiC synthesis, laying the foundation for future optimization of ChiC production and cell-free production of similar proteins.

Optimization of expression in Escherichia coli BL21 (DE3), IMAC purification, storage, and preliminary functional characterization of recombinant Chitinase C from Pseudomonas aeruginosa

Haein Kim — 2023-09-05

Chitin is a homopolymer that is found in the abdominal lining and exoskeleton of various insects. Given the increasing environmental pollution due to chemical insecticides used in agriculture, there is great interest in the development of bioinsecticides. Proteins that hydrolyze chitin, like chitinases, have potential in this regard. Pseudomonas aeruginosa PAO1 Chitinase C (ChiC) is a 55 kDa secreted protein, and while the chiC gene has previously been cloned and expressed in Escherichia coli BL21 (DE3), the optimal isopropyl β-D-1- thiogalactopyranoside (IPTG) inducer concentration, incubation time, and temperature for soluble recombinant ChiC (rChiC) expression have yet to be elucidated. We used a factorial experiment to optimize the conditions for soluble rChiC expression, purified soluble rChiC using immobilized metal affinity chromatography (IMAC) with Ni-NTA resin, and conducted a preliminary functional characterization of rChiC using a chitin-binding assay. We found that the soluble protein is optimally expressed under 0.1 mM IPTG induction, 24 hr incubation, and at 20°C. The present study used histidine in place of the more commonly used imidazole for competitive washing and elution in IMAC, illustrating the potential of histidine as a competitive agent for protein purification. Finally, we observed that the purified rChiC has a functional chitin-binding domain, demonstrated by a chitin-resin binding assay. Overall, our study demonstrates the preliminary optimization of expression, purification, and functional characterization of rChiC present in the soluble fraction in E. coli BL21 (DE3). This study reveals potential for future research aimed at further optimization of soluble rChiC and subsequent functional characterization of the chitinolytic domain of rChiC using in vitro and in vivo systems.

Towards the construction of a chitin-binding domain BioBrick®: PCR Amplification of the Pseudomonas aeruginosa chiC chitin-binding domain

Paula Renee Boldut — 2023-09-05

Historically, the chitin-binding domain (CBD) of the PA01 strain of Pseudomonas aeruginosa, Chitinase C (ChiC), has been employed as a protein tag in purification processes. However, there remains a need for CBD tags with new properties, such as increased temperature stability or binding affinity. The CBD of PAO1 ChiC is a novel CBD showing promise for purification assays as it could have the aforementioned properties. This study aimed to design a gene block of the PAO1 ChiC CBD and insert it into an expression vector to confirm CBD functionality separate from its native protein and its potential as a tag. This gene block was also designed for cloning into a BioBrick® vector, a standardised vector for interchangeable parts. Insertion into a BioBrick® would allow a standardised protocol for tagging proteins with a CBD, through different combinations of BioBrick® parts. Furthermore, because the cysteine residues within the CBDs of other organisms have been critical to proper domain folding, the functional relevance of the sole ChiC CBD cysteine in PAO1 was also explored. To do this, a second gene block was designed with the substitution of alanine for cysteine. This study describes the experimental design for cloning a CBD-encoded gene block into a pET-28a(+) plasmid, evaluating its functional activity using a chitin-binding assay and then cloning the gene block upon functional activity verification into a BioBrick®. In this study, we were able to design these gene constructs and experimentally showed two primers that could amplify the gene construct. These findings have promising implications for the future as the gene constructs could be inserted into a vector. There are then implications for researchers to use this CBD in the assembly of unique protein sequences which could have applications in producing chitin-based materials for use in biomedicine and agriculture.

Optimization of Chitinase C expression from the pM3CRYY plasmid in Escherichia coli BL21(DE3) and determination of secretion status

Jinny Choi — 2023-09-05

Chitin is an essential polysaccharide found in fungal cell walls, crustacean shells, and insect exoskeletons that can be broken down by chitin-degrading enzymes called chitinases. As conventional pesticides contain chemicals associated with a number of severe health risks, chitinase C (ChiC) expressed from the soil microbe Pseudomonas aeruginosa has been proposed as a safer and more sustainable alternative to target chitin-containing pests. A previous study generated a ChiC expression plasmid, pM3CRYY, by cloning chiC from P. aeruginosa PAO1 into a pET28a backbone. Through induction with isopropyl β-D-1-thiogalactopyranoside (IPTG), they demonstrated expression of a 55 kDa protein from pM3CRYY, though the identity of this protein was not confirmed. It is also unknown if the expressed protein is secreted when expressed in E. coli BL21. Thus, we aimed to confirm the identity of 6xHis-tag conjugated ChiC off pM3CRYY, and to determine the optimal IPTG induction conditions for ChiC expression. We found that chiC in pM3CRYY is conjugated to a 6xHis-tag at the 5’ region, and confirmed the presence of His-tagged ChiC in IPTG-induced pM3CRYY Escherichia coli BL21(DE3) cultures. Optimal conditions for protein expression were 0.1 mM IPTG for >6 hours at 37ºC. Additionally, lysing cells by bead beating resulted in higher amounts of protein extracted compared to a boiling lysis method. Interestingly, we also detected 6xHis-tagged ChiC in the soluble fractions of induced cultures. However, it was unclear if the protein was being released into the extracellular space because of active secretion or through the action of cells lysing and releasing their intracellular contents. Therefore, we attempted to determine if ChiC was being secreted under induction conditions by probing for the presence of the putative cytosolic chaperone protein, DnaK. Although our results suggest cytosolic proteins and ChiC are released into culture supernatants as a result of cell lysis, this experiment should be repeated before concluding the secretion status of ChiC in this expression system.

Investigating the chitin-binding capability of recombinant ChiC expressed in E. coli

Leon Lin — 2023-09-05

The chitinase C protein (ChiC), natively expressed by Pseudomonas aeruginosa PAO1, has been explored as a safe and environmentally-friendly crop protection agent. Previous work has been conducted to clone the chiC gene into pET-28a to create pM3CRYY A3, an inducible expression vector for ChiC with an N-terminal 6xHis-tag for purification. In this study, we expressed ChiC in Escherichia coli BL21 (DE3) transformed with the plasmid and purified the expressed protein using nickel-resin immobilized metal affinity chromatography. We then assessed chitin-binding ability using a chitin resin and showed that recombinant ChiC expressed in E. coli can bind to chitin in vitro. Our findings enable further research on the activity, stability, and applications of recombinant chitinases.

Purification of Chitinase C in transformed E. coli BL21 (DE3) preserves a functional Chitin-binding Domain

Cathryn Guo — 2023-09-05

Conventional pesticides play an integral role in commercial agriculture, however, many of these pesticides also have a negative impact on human health, leading to increased interest in finding safe alternatives. Chitin, a biopolymer commonly found in the exoskeleton of insects, has received significant attention as a potential target for the development of biopesticides. Chitinolytic enzymes are naturally produced by many bacterial species such as Pseudomonas aeruginosa. In particular, the 55kDa chitinolytic enzyme chitinase C (ChiC), secreted by Pseudomonas aeruginosa PAO1, has demonstrated insecticidal activities in previous studies. ChiC has been extensively characterized in its native host and previously transformed into other bacteria such as Escherichia coli using the pM3CRYY chiC expression plasmid to isolate ChiC for purification and mechanistic studies. We hypothesized that soluble protein purification of ChiC would allow for the preservation of a functional chitin-binding domain. Purification was performed using immobilized metal affinity chromatography and dialysis, and the activity of the chitin-binding domain in purified ChiC was tested using chitin resin affinity purification. The successful purification of ChiC with a functional chitin-binding domain highlights the potential of the ChiC expression system in E. coli BL21(DE3), which may be further investigated to elucidate the possible functionality of the whole protein.

Refolding of Recombinant Chitinase C of Pseudomonas aeruginosa purified from insoluble fractions using IMAC purification

Soroush Mohebat — 2023-09-05

Chitinase C (ChiC) from Pseudomonas aeruginosa can potentially be developed into a bioinsecticide agent, as it can break down chitin, a crucial component of most insect exoskeletons. However, due to the pathogenic potential of P. aeruginosa, developing ChiC for use in agricultural industries is not desirable. To express recombinant ChiC (rChiC), previous studies transformed Escherichia coli BL21 with pM3CRYY, a pET-28ɑ plasmid containing the chiC gene. Although they observed the expression of rChiC in E. coli, they did not go further to purify rChiC. The aim of this study was to obtain purified and functional rChiC. We investigated the yield of rChiC expression under different sets of conditions in E. coli BL21 transformed with pM3CRYY, followed by purification and refolding of rChiC. The conditions that led to the highest yield of expressed rChiC were found to be 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) concentration with a 2 hrs induction period at 37 °C. At these conditions, the majority of the expressed rChiC was insoluble. The rChiC was purified from the insoluble fraction of the cell lysates using immobilized metal (Ni²⁺) affinity chromatography under denaturing conditions. Subsequently, we refolded the enzyme through dialysis, which yielded 11.7 g of rChiC per 1 L of E. coli culture. The unique pattern of cleavage in limited proteolysis demonstrated that the purified rChiC was refolded into a uniform tertiary structure in dialysis. The successful purification of the refolded rChiC using a chitin-binding column suggested that the enzyme binds to the chitin beads. This suggests that the binding domain of the refolded rChiC is functional. The overall results indicate functional rChiC can be extracted from insoluble fraction of cell lysates with a high yield in a time and cost-efficient manner, which paves the way for future biotechnological applications of ChiC in agricultural industries.

Increased pathological severity of Familial Dysautonomia enriches murine gut microbial composition

Ashleen Khatra — 2023-09-06

Familial dysautonomia (FD) is the most prevalent type of Hereditary Sensory and Autonomic Neuropathies, which are rare genetic neurological disorders that affect both the peripheral and central nervous systems. Previous studies have found significant alterations in the gut microbiome and metabolome in FD patients compared to healthy individuals. However, it is unclear how gut microbial composition differs with varying levels of FD severity. Here, we aim to investigate whether there are compositional differences among murine gut models with mild, moderate, and severe FD. First, we showed a significant increase in species richness as severity increases. Moreover, beta diversity analysis indicated increasing compositional differences as severity increases. Second, a differential abundance analysis revealed significant downregulation of core commensal microbes in mice with severe FD compared to healthy controls. Additionally, Indicator Species Analysis unveiled the presence of unique pathogenic species, such as select Clostridium spp., underlining the relationship between specific bacterial species and FD pathologies. Overall, our findings suggest that increases in gut microbial richness observed in murine models with severe FD is due to the downregulation of some commensal microbes and the introduction of unique pathogenic species. These results accentuate the gut-brain and gut-metabolism axes as promising therapeutic targets for FD.

Non-breast milk diet increases gut microbial diversity and inflammation in six-month-old infants

Soroush Mohebat — 2023-09-08

The interplay between the gut microbiome, inflammation, and diet is established. Previous research has explored the connections between the gut microbiome and inflammation. However, there is a lack of research evaluating this interplay within anemic and non anemic infant populations. This study investigates how anemia and diet, specifically breast milk (BM) and non-breast milk (non-BM) diets (lacking breast milk), affect inflammation and the gut microbiome. At first we found that diet plays a significant role on gut microbial diversity and inflammation, with a non-BM diet showing a significant increase in both gut microbial diversity and inflammation level. We then found that for BM and non-BM diets, anemic status of six-month-old infants does not significantly alter inflammation and the gut microbiota. Further analysis of the core microbiome then showed a higher number of indicator species in the gut microbiome of infants on a non-BM diet and those with elevated inflammation levels. An indicator species analysis of the gut microbiota revealed the presence of potentially pro-inflammatory microbes that were common in infants on the non-BM diet and those with elevated inflammation. These data suggest that a non-BM diet can lead to an increased risk of inflammatory diseases due to an increase in abundance of proinflammatory microbes. Overall, this study highlights the importance of using breast milk when considering an infants' diet, especially in situations where they are exposed to potentially higher inflammation levels.

Increase in gut microbiota taxonomic sensitivity to inflammation from 6 to 12 month infants

Mari Aiko Job — 2023-09-05

Inflammation is a biological response typically indicative of a diseased state, and long-term inflammation is also potentially pathological. Recent studies have highlighted the important relationship between the gut microbiota and inflammation in the human body, identifying a potential avenue of new therapeutics. However, this relationship remains to be comprehensively explored in infants. As such, the objective of this study is to investigate the relationship between gut microbial composition and inflammation in 6 and 12 month old infants. In this study, inflammation markers C-reactive protein and α1-acid glycoprotein were used to examine the overall diversity differences as well as taxonomic changes between infants with high and low inflammation levels. It was found that overall diversity of infants is not significantly impacted by inflammation levels in both 6 and 12 month old infants. By contrast, taxonomic changes reflect an increasing association of specific taxa with inflammation levels as infants age from 6 to 12 months old. These results suggest a potential age-related correlation between the gut microbiome of infants and inflammation status. Overall, this study demonstrates the need to pursue further research in this area to allow for the development of potential early diagnostic tools vital for early intervention and treatment for inflammation in infants.

Iron-deficiency anemia is associated with loss of gut microbial diversity in 6- and 12-month-old infants

Luiza Lopes Pontual — 2023-09-05

Iron-deficiency anemia (IDA) is the most common form of anemia, arising from an inadequate quantity of iron in the body. IDA is especially concerning for young children, as it is linked to neurological dysfunction, immunological deficiencies, and increased mortality. As early life is associated with changes in the human microbiome, we aimed to investigate the effects of IDA on the gut microbiome of 6- and 12-month-old infants. We used a previously generated dataset of 16S rRNA gene sequences to compare diversity metrics, core microbiome analyses, and indicator taxa between IDA and non-IDA reference infants of both age groups. We found that both 6- and 12-month-old infants with IDA had significantly lower microbiome diversities compared to controls. Additionally, the indicator taxa differed between 6- and 12-month-old IDA infants and reference infants. This may suggest IDA could have differential, age-associated impacts on infants. Loss of gut microbial diversity in infants with IDA offers a potential explanation for the cognitive deficits associated with the condition. However, the nature of this relationship remains to be further clarified.

High-fish intake is associated with later-onset Parkinson’s disease and enrichment of short-chain fatty acid-producing microorganisms

Ishana Lodhia — 2023-09-05

Epidemiological studies suggest that dietary habits can modulate the pathogenesis of Parkinson’s disease. As an important source of omega-3 fatty acids, consumption of fish promotes brain health, potentially mediated in part by the microbiome. In this study, we investigated the influence of a high-fish diet on Parkinson’s disease onset and whether this is associated with enrichment of microbial taxa that maintain a healthy gut. We hypothesized that later-onset Parkinson’s disease is correlated with individuals consuming high dietary fish, and that the putative effect is mediated through enrichment of short-chain fatty acid-producing microorganisms. We used dietary information and taxonomic data obtained from fecal samples of 197 Parkinson’s disease patients surveyed by Cirstea et al. We observed a significant correlation between high-fish intake and later-onset Parkinson’s disease in males. Although we did not find global perturbations in gut microbiota composition due to fish intake and age of onset, short-chain fatty acid-producing microbes, including Intestinimonas and Lachnospiraceae, were enriched in high-fish-consuming male Parkinson’s disease patients relative to low-fish. Further, these microorganisms were identified as indicator species for high-fish intake and later-onset Parkinson’s disease. Additionally, we determined that high-fish diets may be associated with short-chain fatty acid metabolic pathways, namely acetate and acetyl-CoA synthesis. Together, our findings suggest that a high-fish diet selects for enrichment of short chain fatty acid-producing microorganisms that may contribute to delaying onset of Parkinson’s disease in males by maintaining gut health.

The gut microbiome belonging to Colombian adults of younger generations has shifted to a more westernized composition compared to older generations

Grace Bodykevich — 2023-09-05

Communities undergoing westernization experience changes to their lifestyle and diet. These changes are associated with decreased levels of activity, increased levels of obesity, increased risk for cardiometabolic disease, and changes to the microbiome composition. Obese individuals tend to have reduced levels of diversity and increased abundance of potentially pathogenic bacteria. Additionally, aging has been associated with increased levels of diversity, which can be linked to changes in lifestyle. In this study, we examined the impact of obesity, defined by waist circumference and body fat percentage, on the gut microbiome of individuals in the middle of westernization. Our study also explored the relationship between gut microbiome composition and obesity across multiple generations. Within our dataset, there was no significant difference in alpha diversity between obese and healthy individuals but there was a significant difference in diversity between two age groups, 18-28 and 38-48. Taxonomic analysis revealed that the age group 18-28 was defined by taxa Bifidobacterium and Bacteroides which are marker taxa for a westernized microbiome, and the age group 38-48 was defined by upregulation of Prevotella, a marker taxa for a non-westernized diet. Taken together, our results indicate that our defined obesity metrics do not have an impact on the gut microbiome of individuals in the midst of westernization. Our results instead highlight the possibility that younger generations may have been more affected by westernization than older generations. Our study has the potential to add to our growing understanding of westernization and its varying impacts across generations.

Individuals with abnormal cardiometabolic statuses are more vulnerable to smoking-induced alterations of the gut microbiome in a Colombian population undergoing Westernization

Ali Reza Nasseri — 2023-09-05

The “Western” diet and lifestyle is of increasing scientific interest due to its rising global prevalence and potential implications for human health. It is associated with a decline in gut microbiome diversity and an abnormal cardiometabolic status, which contribute to inflammatory responses and the development of non-communicable diseases. Additionally, smoking has been implicated in increased morbidity and mortality from cardiovascular diseases and metabolic disorders. However, interactions between smoking, cardiometabolic status, and the gut microbiome have not been previously characterized in the context of Westernization. In this study, we investigated the impact of smoking on the gut microbiome across westernized Colombian individuals with either healthy or abnormal cardiometabolic statuses which were collected during a study performed by Cuesta-Zuluaga et al. For those with an abnormal cardiometabolic status, smoking was associated with reduced microbial species richness, particularly distinct in 8 core microbiome members, including the commensal genera of Bifidobacteria, Christensenellaceae, and Blautia. For westernized individuals with either a healthy or abnormal cardiometabolic status, smoking was associated with a reduced relative abundance of bacterial genera. However, the number of genera experiencing a decrease in relative abundance was greater in individuals with abnormal cardiometabolic statuses. The decrease in relative abundance was represented primarily in the Firmicutes phylum. Furthermore, abnormal cardiometabolic status was associated with potentially harmful bacterial species, such as Desulfovibrio piger and Bacteroides ovatus, which was significantly differentially abundant in smokers and are also prevalent in inflammatory bowel disease patients. Our findings indicate that individuals with abnormal cardiometabolic statuses are more susceptible to smoking-induced changes of their gut microbiomes.

Dietary fiber consumption is associated with the selection of key microbial species but does not affect overall microbial diversity in a Colombian cohort

Jinny Choi — 2023-09-05

The gut microbiome is known to be affected by factors in the host diet. In particular, dietary fiber intake is associated with changes to gut microbial diversity, as one of the key functions of the microbiota is to degrade non-digestible fibers into nutrients and beneficial compounds such as short chain fatty acids (SCFAs). These changes have been characterized in populations at fiber intake extremes, such as populations following Westernized or hunter-gatherer (non-Westernized) lifestyles. However, this relationship has not been well-studied in populations undergoing a shift from a hunter-gatherer to a Westernized lifestyle, a phenomenon currently underway in Colombia. To understand the impact of dietary fiber intake on the host gut microbiota, we analyzed an existing dataset from a Colombian cross-sectional study using a bioinformatics approach. We found that overall microbial diversity does not seem to be impacted by fiber intake. However, there was evidence of over-representation of certain anti-inflammatory SCFA-producing microbial species in individuals with adequate fiber intake. Indicator species analysis also revealed that the enrichment of several SCFA-producing microbes were indicative of adequate fiber consumption. Overall, our findings indicate that dietary fiber does not influence microbial diversity but does enrich specific beneficial microbial taxa in the Colombian population. These results appear to support a beneficial role of fiber consumption and could ultimately aid in our understanding of the gut microbiome in semi-Westernized populations such as the Colombian people.

Exploring non-WEIRD populations gut microbiota: the relative importance of obesity metrics on gut microbiome diversity and composition in Colombian adults

Ghazaleh Shoaib — 2023-09-05

Previously, gut microbiome research has predominantly focused on Western, educated, industrialized, rich and democratic (WEIRD) societies, which limits the generalizability and identification of patterns across ethnicities. This study explores the link between obesity and microbiome variation in an ethnically representative group, which is compared with other global datasets. In this study, we investigated the effect of obesity on the gut microbiome using a dataset with 442 Colombian adults to provide a valuable example of diversity in research. We first analyzed the alpha and beta diversity of obesity-related and general factors. This allowed us to see which ones significantly affected gut microbiota. This revealed some individual obesity-linked predictors affected variation in composition and diversity. However, when the dataset was filtered into obese and non-obese individuals, no specific microbial community compositional differences were found. Despite this, core microbiome analysis revealed certain gut bacterial species were consistently found in obese or non-obese groups. Finally, using model selection to contextualize obesity-related metrics among other predictors, we found that some obesity metrics significantly explained diversity but not composition. This study suggests that although there may be a significant link between obesity and gut microbial variation in WEIRD populations, the patterns may be potentially different in non-WEIRD populations such as Colombian adults.

Core microbiomes found in habitats with sandy bottom substratum are the most phylogenetically diverse among trophic level three ray-finned fish midgut samples

Shutong Gang — 2023-09-08

Fish gut microbiomes play an indispensable role in regulating fish growth, behavior and immune health, yet little is known about the environmental factors that influence its composition. We investigated how habitat substrata affects the midgut microbiome in carnivorous ray-finned fish, hypothesizing that fish from similar substrata have similar midgut microbial compositions. Using alpha diversity metrics, midgut samples demonstrate the highest variety and abundance of microbial species compared to other body sampling sites. Midgut samples from fish found in habitats with kelp forest, sandy bottom and rocky reef substrata exhibit distinct core microbiomes, only sharing one common core species: Pseudoalteromonas sp. In addition, we characterized the relative and total abundance of midgut samples at a phylum level and identified Proteobacteria, followed by Bacteroidetes and Firmicutes, to be the most dominant phyla in the core midgut microbiota. The midgut microbiomes from fish living in habitats with sandy bottom substratum are characterized by the key indicator species Synechococcus sp. CC9902 and Psychromonas sp. Overall, this study demonstrates that the fish gut microbiome is associated with the fish’s habitat, with the key species identified providing the foundation in understanding the major phyla associated with carnivorous fish microbiota. We hope to extend this knowledge into developing probiotics to promote fish digestive and immune health, as well as technology to monitor fish populations.