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Vesicles in the Study of Flaviviruses

July 6, 2023

By Nick Puso, Biochemistry & Molecular Biology ’23

Author’s Note: Nick Puso is a biochemistry & molecular biology graduate of the class of 2023. He wrote this review because, in his own words, he “really loves vesicles”. Nick found this topic particularly exciting to write on because it combines biochemistry, structural biology, genetics, drug design, and lipidomics. He is currently applying to medical school and hopes to become an ER doctor so he can put his love of the biological sciences to good use. Academics aside, Nick loves weightlifting, cycling, backpacking, and pushing wheelchairs at the VA where he volunteers. If you’re looking for him, your best bet is to comb the Sierra Nevadas in a helicopter.

INTRODUCTION 

Flaviviruses, including ZIKA, Dengue virus, and others infect over 400 million people annually [1], causing serious neurological symptoms, hemorrhaging, and birth defects. This issue is exacerbated by the failure of past research to produce any antivirals that prove safe and effective in early clinical trials [2]. An effective antiviral would significantly reduce the global mortality and morbidity of flaviviruses, but development is long and difficult. The process of producing an antiviral relies on knowledge of a critical mechanism in a virus’s infectious biology. In the case that this knowledge applies to more than one virus, the potential exists to develop an antiviral with broad efficacy- saving essential time and money. Flaviviruses as a genus share many fundamental attributes in their infectious biology, particularly concerning their use of vesicles. These conserved features could be exploited to create antivirals with broad efficacy within the genus. The flaviviruses referred to here include Zika (ZIKV), Dengue virus (DENV), Langat virus (LGTV), West Nile virus (WNV), and Powassan virus (POWV). This review will discuss recent advancements in our understanding of the role vesicles play in the infectious biology of flaviviruses. This will include release from endosomes after entry into the cell, viral replication within the unique “vesicle packet” organelle, and a novel exosome-based mechanism of infection. Furthermore, we will explore how this knowledge could inform drug development. 

Background 

Flaviviruses are a genus of enveloped viruses, meaning that their genetic material is contained within a lipid bilayer. Embedded in this bilayer are the Envelope (E) protein (sometimes called E-glycoprotein) and Membrane (M) protein. E-Protein is responsible for attaching naked viral particles (virions) to target cell membranes, upon which they enter the cell via clathrin-mediated endocytosis [3]. The clathrin coated proteins mold a small bubble of host membrane material–an endosomal vesicle–which carries the virions into the cell.

For the viral genome to escape the endosome, and enter the cytoplasm, the viral envelope must fuse with the membrane of the endosome. Fusion is initiated by the acidic pH of endosomes, which drives a conformational shift in viral E-protein that merges the viral envelope with the endosome [3]. This mechanism is conserved in all flaviviruses. 

Flaviviruses also utilize a unique replication organelle, derived from the membrane of the endoplasmic reticulum (ER), called the vesicle packet. The vesicle packet is an invagination into the ER membrane in which the machinery of viral replication is contained, and likely protected from host cell innate-immune factors. A combination of host factors and viral proteins, including non-structural protein 1 (NS1) conserved in flaviviruses, work together to construct the vesicle. [4]. However, the specific host factors involved in vesicle packet formation were largely unknown until recently. 

Once translated and assembled, virions exit the cell via the secretory pathway, including the trans-Golgi-network (TGN). Like the endosome earlier in the life cycle, the TGN has an acidic pH. M-protein requires this acidic pH to mature. Specifically, prM, the precursor to M-protein, undergoes an acid-driven conformational shift which exposes cleavage sites for Furin-protease, a host protease [5]. Cleavage releases mature M-protein from ‘prM’. The ‘pr’ element itself is responsible for preventing premature fusion of the virion envelope with host membranes, so it remains associated with the virion until secretion [5]. The dissociation of ‘pr’ after cleavage allows the mature virion to later undergo acid-driven fusion with endosomes in a new cell, completing the cycle [5]. 

Endosomal release and niclosamide’s antiviral potential 

A compound that safely inhibits endosomal and secretory pathway acidification could represent a pan-flavivirus antiviral. This is because attenuation of acidic pH would prevent the conformational shift in E-protein necessary for the fusion of the virion with the endosome. Recently, researchers determined that niclosamide–an FDA-approved antiparasitic drug previously known to inhibit ZIKV infection–did exactly this in DENV-infected cells [6]. Niclosamide is a protonophore;a compound that can exchange protons across membranes, bypassing the proton pumps responsible for endosomal acidification. Treatment with niclosamide significantly reduced viral load in baby-hamster-kidney (BHK21) cultures infected with DENV. Using pH-sensitive dye A0, which turns from clear to pink at pH 6, the mechanism was confirmed to be attenuation of endosomal acidification. Additionally, a western blot of DENV protein-E showed that the acid-driven confirmation shift in protein-E necessary for endosomal fusion did not take place. This western blot relied on the fact that E-protein’s conformational shift results in its degradation. An antibody could visualize E-protein in the SDS-page (electrophoresis which separates proteins by mass) of the treatment group, meaning the degradation did not take place. This indicates that the conformational shift was blocked by niclosamide as hypothesized. Because no effects were noted on viral genome replication or endocytosis independently of endosomal release, the authors do not put forward any additional mechanisms of action. 

However, another study found that niclosamide also prevents the maturation of DENV in human (Huh7) cells via deacidification of the TGN [7]. Researchers determined that deacidification inhibited cleavage of DENV/ZIKV prM protein by preventing the exposure of furan-protease cleavage sites. Compared to a control, Western blot of the treatment grouprevealed uncleaved prM when niclosamide was present. Virions with immature prM protein due to niclosamide treatment would be unable to fuse with endosomes upon infection of a new cell and are thus dead. The authors suggest discrepancies from Kao et al [6] arrose because they used different time points in their analysis of niclosamide’s effect on genome replication and virion maturation. 

Niclosamide’s ability to de-acidify endosomes and the TGNdemonstrates its potential as a pan-flavivirus antiviral, given that E-protein and prM are conserved and essential in all flaviviruses. Both authors argue that niclosamide’s safety as an antiparasitic drug and its effect on ZIKA and DENV warrant investigation into antiviral applications. Additionally, both studies put forward inhibition of endosomal release as a viable strategy in anti-flavivirus drug development overall. Jung et al also recommend inhibition of prM cleavage for therapeutic investigation.

Targeting host factors in ‘Vesicle packet’ formation 

Replication inside the vesicle packet is another potential target for pan-flavivirus antiviral development because it is conserved in the genus. Knowledge of the involved host factors–proteins in the host that the virus manipulates to its own ends–could allow for antivirals that knockout vesicle packet formation, inhibiting viral replication. Recently, a class of host ER-shaping proteins called Atlastins were found to play a role in the formation of the vesicle packet [8]. Researchers used small-hairpin RNAs (shRNAs), which are processed into silencing siRNA to knock out various atlastins in A529 cells exposed to ZIVK, DENV, and WNV. Transmission electron microscopy, which uses electrons to visualize microscopic structures, revealed that Atlastin-2 (ATL2) knockout shrunk both the size and number of vesicle packets, and disrupted their localization. Immunofluorescence, which visualizes the location of small molecules using a fluorescent antibody, then showed that the localization of viral double-stranded RNA (dsRNA) was changed. This indicates that the coordination of vesicle packets with the replicating viral genome was disrupted. Also, the overall viral load of ZIKV, DENV, and WNV was reduced by Atlastin-2 knockout. In the case of ZIKV, this reduction was potentially as much as 16-fold. While the authors make no specific recommendations, future research should explore whether these factors inhibit flavivirus replication outside of DENV, ZIKV, and WNV. Given that all flaviviruses form a vesicle packet for replication, the host factors they manipulate to do so may be the same. If this is the case, disruption of flavivirus/Atlastin interaction may be a powerful therapeutic approach. 

A host protein called Receptor of Activated C Kinase (RACK1)has also been identified as essential to vesicle packet formation for flaviviruses WNV, DENV, POWV, and LGTV [9]. Researchers used a similar approach to Neufeldt et al, employing small ‘interfering’ RNAs (siRNAs) to silence host factors and measure the effect on viral load. Specifically, a CRISPR-based genome-wide knockout screen using LentiCRISPRv2-GecKO (an siRNA library) provided siRNAs to human Huh7.5 cells. RACK1 knockout produced the strongest reduction in viral load out of all targets. To determine the mechanism, researchers analyzed the time dependency of viral load reduction on RACK1 knockout. The results indicated that RACK1 did not act on viral entry or translation, but rather on replication, which brought attention to the vesicle packet. Transmission electron microscopy then demonstrated a significant reduction in vesicle packets after RACK1 knockout. Immunofluorescence found that viral NS1, a known critical factor in vesicle packet formation, failed to localize to the ER. The failure of NS1 to localize to the ER during RACK1 knockout would suggest that an interaction between NS1 and RACK1 is necessary for vesicle packet formation. Co-expression of RACK1 and NS1 gave strong evidence for their co-localization. To confirm the interaction, a pulldown assay- which measures binding affinity between two molecules- was performed and RACK1 was proven to bind NS1. The authors thus posit that RACK1 is responsible for localizing NS1 and that RACK1 knockout disrupts vesicle packet formation by the failure of NS1 to localize. 

The discovery of ATL2 and RACK1 as host factors necessary to the formation of the vesicle packet offers new targets for disrupting flavivirus replication. The respective authors argue that dependence on RACK1 and Atlastin-2 for vesicle packet formation is likely conserved in all flaviviruses. Shue et al thus argue that RACK1 is a promising target for antiviral development, and recommend further research into RACK1 knockout as a therapeutic strategy. ATL2’s importance to vesicle packet formation warrants exploration into its potential as a pan-flavivirus antiviral target. 

Exosome-based infection: SMase and Tsp29Fb as drug targets 

Recently, three studies have identified a novel exosome-based mechanism of flavivirus infection and identified potential targets for antiviral development. Zhou et al, (2018) found that LGTV uses extracellular vesicles (exosomes) from its tick host to infect mammalian cells, and that infected mammalian brain-endothelial cells, which make up the blood-brain barrier, produce exosomes capable of infecting neuronal cells [10]. In a follow-up to their 2018 study, Zhou et al (2019) found that ZIKA-infected, neuronal-cell-derived exosomes readily infected neurons of the cortex [11]. A third study, Vora et al, demonstrated that exosomes derived from DENV-infected mosquitoes were infectious to human blood-endothelial cells [12]. The ability of flaviviruses to use exosomes, derived from host cells, to infect other cells was previously unknown and represents a significant advancement in our understanding of their infectious biology as a genus. 

Additionally, GW4869, an exosome release inhibitor, proved to reduce viral load in cell cultures infected with their respective flavivirus in all cases. GW4869 works by inhibiting sphingomyelinase (SMase), an enzyme 

that produces lipids necessary for exosome formation. The effect of GW4869 on viral load confirms that exosomes provide significant infectious potential, in a novel mechanism, for 3 prominent flaviviruses: LGTV, ZIKV, and DENV. GW4869’s ability to inhibit this mechanism in all three suggests that an antiviral targeting exosome release may have efficacy across the genus. All three studies thus cite GW4869 as a promising target for drug development to disrupt flavivirus infection via exosomes. GW4869’s efficacy also suggests the anti-flaviviral potential of SMase inhibition more broadly. 

All three studies used immunoblotting and quantitative real-time PCR to show the enrichment of viral RNA and proteins in infectious exosomes.

All three also confirmed that these viral components are fully inside the exosomes. To do this, they exposed these exosomes to RNase and flavivirus envelope protein antibodies, which neutralize the infectious potential of naked virions by degrading exposed viral RNA and protein. The infectious potential of exosomes was unaffected by RNase or antibody treatment. All three studies thus argue that the exosome protects viral RNA and protein from antibodies and RNase. 

Vora et al also found that Tsp29Fb–an ortholog of high similarity to human exosome marker CD63–was enriched on infected extracellular vesicles [12]. Specifically, qRT-PCR, which tracks the expression of RNA over time, found that Tsp29Fb was overexpressed during infection. Additionally, the CD63 antibody (which is highly cross-reactive with Tsp29Fb) was used in immunoblotting to show that Tsp29Fb is enriched on the exosomes. Researchers then used immunoprecipitation, which removes a target from solution using an antibody, to find that Tsp29Fb binds DENV envelope protein. This binding interaction indicates Tsp29Fb may play a role in DENV’s ability to use exosomes for infection. To test this, researchers silenced Tsp29Fb with siRNA and found drastically reduced viral protein in the resulting exosomes. The researchers posit that Tsp29Fb thus plays a role in loading protein into secretory vesicles post-translation, which then exit the cell as infectious exosomes. For that reason, the authors put Tsp29Fb silencing forward as an antiviral development tactic.

These three studies demonstrate that extracellular vesicles provide infectious potential to three flaviviruses. Additionally, two promising candidates for antiviral research have been put forward to disrupt viral exosomes. Namely, GW4869 targeting SMase, and Tsp29Fb as a target itself. 

CONCLUSION

Flaviviruses as a genus have great similarities in their infectious biology concerning their manipulation of host membranes and vesicles. Post-entry endosomal release and the formation of the vesicle packet are shared features of the flavivirus infection cycle. In a completely new infectious mechanism, LGTV, ZIKV, and DENV were all found to invade new cells using exosomes. All three discussed uses of vesicles have been proven essential to infection and are present in multiple flaviviruses. Furthermore, workable targets for the inhibition of these faculties have been identified, which subsequently reduce viral load in vitro. Multiple promising candidates for drug development have thus emerged from these targets. Future research should focus on whether an exosome-based infection is present in other flaviviruses and establish the relevance of SMase and Tsp29Fb to the broader genus. If exosome-based infection proves critical to flaviviruses more broadly, SMase may be a viable target, and GW4869 might have potential as a pan-flavivirus antiviral. With respect to targeting replication in the vesicle packet, RACK1 and ATL2 knockout should be investigated for safety and efficacy. Niclosamide’s ability to inhibit post-entry edosomal release must also be established in the genus as a whole, and the therapeutic dose already approved by the FDA for other conditions should be tested for efficacy against flavivirus.

REFERENCES

  1. Pierson, T.C., Diamond, M.S. The continued threat of emerging flaviviruses. Nat Microbiol 5, 796–812 (2020). https://doi.org/10.1038/s41564-020-0714-0
  2. Qian, Qi. 2022. Mosquito-Borne Flaviviruses and Current Therapeutic Advances. Viruses. 2022, 14(6), 1226; https://doi.org/10.3390/v14061226
  3. Modis Y, Nayak V. Molecular Mechanisms of Flaviviral Membrane Fusion. J Virol. 2009:265–86. doi: https://doi.org/10.1007%2F978-0-387-79840-0_12 
  4. Chatel-Chait, Bartesenschlager. 2014. Dengue Virus- and Hepatitis C Virus-Induced Replication and Assembly Compartments: the Enemy Inside—Caught in the Web. Virology. 2014; 88(11). doi: https://doi.org/10.1128/jvi.03404-13
  5. Yu et al. 2009. Association of the pr Peptides with Dengue Virus at Acidic pH Blocks Membrane Fusion. Virology. 2009 Dec; 83(23): 12101–12107. doi: 10.1128/JVI.01637-09 
  6. Kao JC et al. 2018. The antiparasitic drug niclosamide inhibits dengue virus infection by interfering with endosomal acidification independent of mTOR. PLoS Negl Trop Dis. 2018 Aug 20;12(8):e0006715. doi: https://doi.org/10.1371%2Fjournal.pntd.0006715 
  7. Jung, E. et al. 2019. Neutralization of Acidic Intracellular Vesicles by Niclosamide Inhibits Multiple Steps of the Dengue Virus Life Cycle In Vitro. Sci Rep 9, 8682 (2019). https://doi.org/10.1038/s41598-019-45095-1
  8. Neufeldt et al. 2019. ER-Shaping Atlastin proteins act as central hubs to promote flavivirus replication and virion assembly. Nat Microbiol. 2019 Dec; 4(12): 2416–2429. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881184/
  9. Shue et al. 2021. Genome-wide CRISPR screen identifies RACK1 as a critical host factor for flavivirus replication. Virology. 2021; 95(24). doi: https://doi.org/10.1128/JVI.00596-21
  10. Zhou et al. 2018. Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells. PLoS Pathog. 2018 Jan; 14(1): e1006764. doi: 10.1371/journal.ppat.1006764 
  11. Zhou et al. 2019. Exosomes mediate ZIKA virus transmission through SMPD3 neutral sphingomyelinase in cortical neurons. Emerging Microbes and Infections. PLoS Pathog.2019; 8(1): 307–326. doi:1080/22221751.2019.1578188
  12. Vora et al. 2018. Arthropod EVs mediate dengue virus transmission through interaction with a tetraspanin domain containing glycoprotein Tsp29Fb. Proc Natl Acad Sci USA. 2018 Jul 10; 115(28): E6604–E6613.https://doi.org/10.1073%2Fpnas.1720125115

Review: The role of gut microbiota on Autism Spectrum Disorder (ASD) and clinical implications

November 13, 2021

By Nikita Jignesh Patel, Neurobiology, Physiology, & Behavior ’22

Author’s Note: Ever since I took BIS2C at UC Davis, I was intrigued as to how our gut microbiome plays such a huge role in our homeostasis beyond just digestion – in particular, the correlation between decreased microbiome diversity and allergies we learned about in the lab fascinated me. I recently stumbled upon the term “gut-brain axis” and was in awe as to how this connection between our gut microbes and our brain even exists, and learned that gut microbiome diversity is implicated in a plethora of mental disorders, from depression and anxiety, to autism. I decided to write this review to share my learning of how the gut microbiome can change the brain and potentially contribute to Autism Spectrum Disorder (ASD), because I feel as if this is not a widely known correlation – even as a physiology major, I never learned about the gut-brain axis in my courses. Moreover, the cause of autism is still widely undefined and the gut microbiome may provide a possible explanation for ASD onset in some individuals. I believe a wide range of students will find this upcoming research interesting, but my intended audience is those who research autism or work with autistic individuals, as it may provide an explanation for ASD and seems to be a likely target for clinical therapy for autism in the future. Above all, I want my readers to take away a better understanding of the gut-brain axis and how its imbalance can be implicated in brain disorders like autism.

 

Introduction

Autism Spectrum Disorder (ASD) is a lifelong neurodevelopmental disorder characterized by a range of symptoms including difficulty with communication, social interaction, and restricted and repetitive behaviors that present differently in every individual [1]. Although 1 in 54 children are estimated to be on the autism spectrum according to the CDC [2], the etiology of the condition remains poorly understood. Factors including genetics and certain maternal environmental conditions have been identified as potential contributors to the development of ASD in children, but the exact cause is still unknown [3].

 A common comorbidity experienced by ASD individuals is gastrointestinal (GI) problems—including abdominal pain, constipation, and diarrhea —as such, autism research is pivoting towards studying the gut microbiome. [1]. Specifically, a link between the composition of the gut microbiome and brain development has been established in recent years — termed the “gut-brain axis”— and it appears to be the future of autism research. This literature review aims to identify the role of the human gut microbiome on the development of autism-like behavior and investigate whether therapies targeting the gut microbiome can be effective clinical treatments for Autism Spectrum Disorder (ASD). The article will first define the differences observed in gut microbiota between autistic and neurotypical individuals, then discuss how these differences in composition may affect brain development, and finally propose clinical implications targeting gut microbiota that appear promising in the treatment and diagnosis of ASD-related behaviors.  

Gut Microbiome of ASD Patients Differ From Neurotypical Individuals

The gut microbiomes of Autism Spectrum Disorder (ASD) patients have defining characteristics that significantly differ from those of neurotypical individuals. The human gut microbiome consists of a diverse array of predominantly bacteria but also archaea, eukarya and viruses that possess unique microbial enzymes to aid humans in digestion and also a variety of other physiological functions [4]. The three phyla Firmicutes, Bacteroidetes, and Actinobacteria [5] encompass the majority of bacteria present in the gastrointestinal (GI) tract that aid in these functions. However, an imbalance between the ratio of Firmicutes to Bacteroidetes bacteria is found in autistic individuals when compared to the microbiome composition of neurotypical subjects; in particular, patients with autism tend to have an overexpression of Firmicutes in their gut [6,7]. Other studies have demonstrated an excess of the Clostridium genus in the ASD microbiome [8] as well as an overexpression of the genus Bacilli in the mouths and gut of autistic individuals [7]. While the cause behind this imbalance is unknown, these findings signify a consistent pattern of microbial imbalance in the autistic gut microbiome. In fact, a Random Forest prediction model, a computer algorithm that can classify large sets of data into subgroup “trees” based on data similarity, was able to distinguish ASD children from neurotypical children with a high degree of certainty from just microbiome sequencing data [7], demonstrating the predictability of this dysbiosis by artificial intelligence. 

Figure 1: This illustrates the difference between species richness and species abundance. Species richness, a measure of alpha diversity, informs on how many species are present in a sample. Species abundance describes how many organisms of each species are present. 

Along with microbial imbalance—termed dysbiosis—autistic children also tend to have a decreased alpha diversity [7,9], which measures mean species diversity, as well as significantly lower gut species richness [7], the number of species present, when compared to age and sex-matched neurotypical children. One study found that for neurotypical children, alpha diversity, species richness, and species abundance all increased between the age groups 2-3 to 7-11; yet for ASD children, no significant development in microbial composition was observed with increase in age [7]. Since autism has been found to slow brain development as children age [9], this reduced development of the microbiome mirrors the altered brain development characteristic of ASD pathophysiology, proposing an association between decreased microbial diversity and autism.

Due to this observed correlation between dysbiosis and ASD, whether gut dysbiosis is truly causal for autism has come into question. In a preliminary study, Sharon et al transplanted fecal microbiota from autistic donors into otherwise germ-free mice (mice with a sterile gut) and observed their offspring’s behavior compared to offspring of mice inoculated with microbiota from neurotypical donors. Notably, mice with the ASD microbiome— characterized by decreased alpha and beta diversity and decreased Bacteroidetes — exhibited behaviors paralleling those of autism, including repetitive behaviors, decreased locomotion and decreased communication [9]. This demonstrated that gut dysbiosis can in fact induce the behavioral deficits observed in ASD. This is significant evidence toward the theory that gut dysbiosis indeed contributes to ASD – an important finding that changes our current understanding of the etiology of autism.

Figure 2: Above is a visual depiction of the study conducted by Sharon & colleagues, where germ-free mice were inoculated with gut microbiota from either autistic or neurotypical donors. Offspring of the mice transplanted with ASD microbiome were shown to exhibit autism-like behavior.

How Microbiota Imbalance Affects Brain Function: The Gut-Brain Axis

Since the microbial dysbiosis found to be common in ASD patients contributes to behavioral deficits, several different mechanisms have been proposed for how the altered microbial environment in ASD patients can affect brain development.

Intestinal permeability

The microbes that line the GI tract provide structural and protective benefits to our intestines, including stimulating epithelial cell regeneration and mucus production by the intestinal walls. When microbial diversity is decreased, the integrity of the intestinal walls may be compromised which can lead to increased intestinal permeability [8]. This may allow for lipopolysaccharide (LPS), a pro-inflammatory endotoxin that is found in gram-negative bacterial cell walls, to escape out of the GI tract and into the bloodstream. Serum levels of LPS are in fact found to be significantly higher in autistic individuals [12]. LPS causes inflammation in the central nervous system (CNS) and is found to impair cognition and motivation in the mouse model. Specifically, implications for impaired continuous attention and curiosity behaviors, along with modulation of other areas of the brain like the central amygdala have been associated with circulating LPS [11]. Therefore, altered intestinal permeability is a possible mechanism by which dysbiosis modulates brain inflammation, a hallmark of autism that is thought to contribute to its behavioral symptoms.  

Microbial metabolites

As gut microbes carry out cellular functions inside their human hosts, they also secrete compounds as by-products of metabolism. Two such metabolites are 5AV and taurine, which are secreted by gut Bacteroides xylanisolvens and other bacteria. 5AV and taurine levels are found to be significantly lower in autistic individuals [13,14] as well as mice transplanted with ASD microbiome [9], likely due to dysbiosis. Both 5AV and taurine are gamma-aminobutyric (GABA) receptor antagonists, meaning that lower levels of these circulating microbial metabolites can alter the inhibitory signaling of GABA in the nervous system [9]. GABA regulates various developmental processes in the brain, including cell differentiation and synapse formation, so dysfunction in GABA signalling is thought to account for ASD symptoms [15]. Oral administration of taurine and 5AV in a mouse model of ASD with an altered microbiome is shown to reduce repetitive behavior and increase social behavior, suggesting that the deficiencies in these metabolites may contribute to the behavioral manifestations of autism [9]. There are other microbial metabolite imbalances in autistic children, including dopaquinone, pyroglutamic acid, and other molecules involved in neurotransmitter production. These imbalances affect brain signaling pathways, and therefore could contribute to the behavioral deficits often present in autistic children. Further, these metabolite imbalances correlate with the levels of certain gut bacteria, further emphasizing the link between the gut microbiome and neurological disorders such as ASD.

Clinical Implications for ASD Diagnosis and Treatment

Today, symptoms of autism are alleviated with behavioral and educational therapy, and no pharmaceutical treatment exists [1]. With the knowledge that the gut microbiome significantly differs in autistic individuals and that these differences are shown to interfere with the nervous system, preliminary research has been done on potential diagnostics and pharmaceutical therapeutics for ASD that target dysbiosis in the gut. 

Diagnostics

To date, there is no objective laboratory test to detect Autism Spectrum Disorder (ASD) in children, so autism is primarily diagnosed through a doctor’s evaluation of a patient’s behavior and developmental history. However, the ability of a computer program to distinguish the autistic microbiome from the neurotypical microbiome holds potential for use in ASD clinical risk assessments through analysis of the gut microbiome, and subsequent gut health monitoring interventions for those detected to have ASD-like dysbiosis [7]. The strong association between the presence of certain bacterial species in the mouth and bacteria in the gut — in particular the significant positive correlation between saliva Chloroflexi and gut Firmicutes—may suggest possible oral biomarkers to predict gut microbial diversity [6]. In addition, the overexpression of certain bacteria in the gut have been identified to be associated with certain symptoms like allergies and abdominal pain, opening an avenue to improve the diagnosis process of ASD through the inclusion of a more objective, laboratory-based test [6].

Microbiota Transfer Therapy

Microbiota Transfer Therapy (MTT) is an emerging therapy that aims to replace the gut microbiome of ASD individuals with a more diverse, healthy gut microbiome. One form of MTT consists of a two-week oral vancomycin antibiotic treatment, followed by a bowel cleanse using MoviPrep, and then finally an extended fecal microbiota transplant for 7-8 weeks, administered orally or rectally. In a clinical trial involving autistic children, MTT significantly increased gut bacterial diversity 8 weeks after treatment stopped, along with improving GI symptoms (including abdominal pain, indigestion, diarrhea and constipation) measured through the Gastrointestinal Symptom Rating Scale (GSRS). Significant improvements in behavioral ASD symptoms were found post treatment as well, measured through increases from baseline scores on a variety of exams that evaluate social skills, irritability, hyperactivity and communication, among other behaviors [16]. These improvements in microbial diversity and subsequently ASD-related behavior were all found to have been maintained at follow-up study two years later, indicating that MTT is a safe and efficient therapy that has potential to improve ASD outcomes in the long-term [17]. However, further studies on the efficacy of MTT are necessary to establish this connection, as the above study sample was small and most symptoms and improvements were self-reported. 

Probiotics

Because imbalances in the microbiome are correlated with autism, direct administration of bacterial cultures using probiotics seems to be a potential approach to treat ASD behavioral symptoms. Probiotic treatment that included a combination of Streptococcus, Lactobacillus, and Bifildobacterium was found to be effective in improving core behavioral symptoms of ASD, specifically adaptive functioning, developmental pathways, and multisensory processing in autistic children with GI symptoms [18]. Probiotics have been shown to improve symptoms of other mood disorders like anxiety and depression, both of which are associated with dysbiosis and the gut-brain axis [8], warranting further research on probiotics as a treatment for ASD. Therapies that target microbial metabolite imbalances in ASD individuals, like 5AV and taurine, may also open an avenue for future autism research [9].

Conclusion

The gut microbiome contributes to the maintenance of much of human physiology, with involvement in not only the digestive system but also the immune system and the brain. Dysbiosis of the gut microbiome has been found to be prevalent in children and adults with Autism Spectrum Disorder (ASD), and this dysbiosis may be linked to the behavioral symptoms observed. Treatments that target the gut microbiome, therefore, serve to be useful in improving behavioral deficits associated with ASD and should be a consideration for future research with more rigorous experimental design.

 

References:

  1. Mayo Clinic. Autism Spectrum Disorder. Accessed July 30, 2021. Available from: https://www.mayoclinic.org/diseases-conditions/autism-spectrum-disorder/symptoms-causes/syc-20352928.
  2. Centers for Disease Control and Prevention. Data & Statistics on Autism Spectrum Disorder. Accessed July 30, 2021. Available from: https://www.cdc.gov/ncbddd/autism/data.html.
  3. Fattorusso A, Genova L, Dell’Isola G, Mencaroni E, Esposito S. 2019. Autism Spectrum Disorders and the gut microbiota. Nutrients.11(2):521. 
  4. Kho Z, Lal S. 2018.The human gut microbiome—A potential controller of wellness and disease. Frontiers in Microbiology. 9:1835.
  5. Thursby E, Juge N. 2017. Introduction to the human gut microbiota. Biochemical Journal. 474(11): 1823-1836.
  6. Kong X, Liu J, Cetinbas M, Sadreyev R, Koh M, Huang H, Adeseye A, He P, Zhu J, Russell H, Hobbie C, Liu K, Onderdonk A. 2019. New and preliminary evidence on altered oral and gut microbiota in individuals with Autism Spectrum Disorder (ASD): Implications for ASD diagnosis and subtyping based on microbial biomarkers. Nutrients. 11(9): 2128
  7. Dan Z, Mao X, Liu Q, Guo M, Zhuang Y, Liu Z, Chen K, Chen J, Xu R, Tang J, Qin L, Gu B, Liu K, Su C, Zhang F, Xia Y, Hu Z, Liu X. 2020. Altered gut microbial profile is associated with abnormal metabolism activity of Autism Spectrum Disorder. Gut Microbes. 11(5): 1246-1267
  8. Mangiola F, Ianiro G, Franceschi F, Fagiuoli S, Gasbarrini G, Gasbarrini, A. 2016. Gut microbiota in autism and mood disorders. World Journal of Gastroenterology. 22(1): 361-368.
  9. Sharon G, Cruz N, Kang D, Gandal M, Wang B, Kim Y, Zink E, Casey C, Taylor B, Lane C, Bramer L, Isern N, Hoyt D, Noecker C, Sweredoski M, Moradian A, Borenstein E, Jansson J, Knight R, Metz T, Lois C, Geschwind D, Krajmalnik-Brown R, Mazmanian S. 2019. Human gut microbiota from Autism Spectrum Disorder promote behavioral symptoms in mice. Cell. 177(6): 1600-1618
  10. Hua X, Thompson P, Leow A, Madsen S, Caplan R, Alger J, O’Neill J, Joshi K, Smalley S, Toga A, Levitt J. 2013. Brain growth rate abnormalities visualized in adolescents with autism. Human Brain Mapping. 34(2):425-36.
  11. Haba R, Shintani N, Onaka Y, Wang H, Takenaga R, Hayata A, Baba A, Hashimoto H. 2012. Lipopolysaccharide affects exploratory behaviors toward novel objects by impairing cognition and/or motivation in mice: Possible role of activation of the central amygdala. Behavioral Brain Research. 228(2):423-31.
  12. Emenuele E, Orsi P, Boso M, Broglia D, Brondino N, Barale F, Ucelli di Nemi S, Politi P. 2010. Low-grade endotoxemia in patients with severe autism. Neuroscience Letters. 471(3):162-5
  13. Ming X, Stein T, Barnes V, Rhodes N, Guo L. 2012. Metabolic perturbance in autism spectrum disorders: a metabolomics study. Journal of Proteome Research. 11(12): 5856-62
  14. Park E, Cohen I, Gonzalez M, Castellano M, Flory M, Jenkins E, Brown W, Schuller-Levis G. 2017. Is taurine a biomarker in Autistic Spectrum Disorder. Advances in Experimental Medicine and Biology. 975
  15. Pizzarelli R, Cherubini E. 2011. Alterations of GABAergic signaling in Autism Spectrum Disorders. Neural Plasticity. 2011:297153
  16. Kang D, Adams J, Gregory A, Borody T, Chittick L, Fasano A, Khoruts A, Geis E, Maldonado J, McDonough-Means S, Pollard E, Roux S, Sadowsky M, Lipson K, Sullivan M, Caporaso J, Brown R. 2017. Microbiota Transfer Therapy alters gut ecosystem and improves gastrointestinal and autism symptoms: an open-label study. Microbiome. 5(1):10
  17. Kang D, Adams J, Coleman D, Pollard E, Maldonado J, McDonough-Means S, Caporaso J, Krajmalnik-Brown, R. 2019. Long-Term benefit of Microbiota Transfer Therapy on autism symptoms and gut microbiota. Scientific Reports. 9(1):5821
  18. Santocchi E, Guiducci L, Prosperi M, Calderoni S, Gaggini M, Apicella F, Tancredi R, Billeci L, Mastromarino P, Grossi E, Gastaldelli A, Morales M, Muratori F. 2020. Effects of probiotic supplementation on gastrointestinal, sensory and core symptoms in Autism Spectrum Disorders: A randomized controlled trial. Frontiers in Psychiatry. 11:550593

Among Virions

August 20, 2021

By Jordan Chen, Biochemical Engineering ‘24

 

What are viruses? Miniscule packages of protein and genetic material, smaller than all but the smallest cells, relatively simple structures on the boundaries of what we consider living. Undetectable to the human eye, these invisible contagions are rarely on the minds of the average person, occupying a semantic space in public consciousness more often than they are understood for their material reality. Stories are more likely to be described as “viral” than an actual virus, yet when the COVID-19 pandemic washed over the world at the end of 2019, the public suddenly had to confront that which was seemingly abiotic, simple, and small. However, the impact of the COVID-19 pandemic exceeded that unassuming material reality. With the shuttering of the global economy, mass death, political crisis, confusion, hysteria, and science without immediate answers, it’s become clear that the sum of COVID-19’s viral components is much more than the whole.

To emphasize this idea in the piece, coronavirus virions are depicted as massive and detailed larger than earth bodies, in a vital bloody red, surrounding and overwhelming the relatively simply shaded globe. What was formerly small, simple, and nonliving, can now be dramatically understood as larger than life, having created complex predicaments, and having taken on a life of its own in its assault against the world. This digital artwork was created in Blender.

Oral Microbiome Imbalances Could Provide Early Warning of Disease

June 18, 2021

Image caption: Fragments of amyloid precursor protein aggregate in β-amyloid plaques, seen here in dark brown. These plaques have been found in the brains of patients with Alzheimer’s disease. Credit: Wikimedia Commons.

 

By Daniel Erenstein, Neurobiology, Physiology & Behavior ‘21

Author’s Note: I first learned about research on the oral microbiome while covering this year’s annual meeting of the American Association for the Advancement of Science in February. Under the theme of “Understanding Dynamic Ecosystems,” the conference, which was held virtually, welcomed scientists, journalists, students, and science enthusiasts for four days of sessions, workshops, and other talks. The human microbiome, home to trillions of bacteria and other microbes, is as dynamic an ecosystem as they come. This article focuses on the bacteria that live in our mouths and their fascinating role in diseases such as diabetes and chronic kidney disease. With this article, I hope that readers consider further reading on the diverse, lively ecosystems within our bodies. For more stories on the microbiome, The Aggie Transcript is a great place to start.

 

There is more to the oral microbiome than meets the mouth. Established within a few minutes of birth, this diverse community of bacteria, fungi, and other microbes lives on every surface of our mouths throughout our lives [1]. For decades, scientists have researched these microorganisms and their role in dental diseases.

But far less is known about the interactions of these bacteria and their products with other parts of the body, and these interactions could hold a particularly important role in human health.

“We’ve always thought of the mouth as somehow in isolation, that oral health does not somehow impact the rest of the body,” said Purnima Kumar, DDS, MS, PhD, professor of periodontology at Ohio State University, during a session at the American Association for the Advancement of Science annual meeting that took place on February 8 [2].

Scientists, though, are increasingly looking to the oral microbiome for answers to questions about health and disease [3].

“The time is absolutely right for us to start putting the mouth back into the body,” Kumar said during the session “Killer Smile: The Link Between the Oral Microbiome and Systemic Disease.”

Panelists highlighted three systemic diseases — diabetes mellitus, rheumatoid arthritis, and Alzheimer’s disease — and their unexpected connection to disturbances in the oral microbiome [4-6]. A common thread running through all three diseases is an association with periodontitis, a gum disease triggered by the accumulation of bacteria, viruses, and even fungi in dental biofilm, or plaque, on the surface of teeth [7].

In health, there is peace between oral microbes and our immune system. Healthy and frequent communication — molecular diplomacy between bacteria and immune surveillance — maintains stable relations. But microbial imbalances due to bacterial buildup in plaque can cause inflammatory immune reactions, resulting in the gradual breakdown of the barrier between biofilm and gum tissue.

“When you have gum disease, that crosstalk, that communication, that harmony is broken down,” Kumar said.

When bacteria subsequently invade our gum tissues, there are consequences for human disease [8].

Mark Ryder, DMD, professor of periodontology at University of California, San Francisco, studies the role of one such bacterium, Porphyromonas gingivalis, in disease [9]. This bacterium secretes enzymes called gingipains, which are essential for its survival.

In the case of Alzheimer’s disease, these gingipains can travel through the bloodstream, cross the blood-brain barrier, and accumulate in brain regions like the hippocampus, which is involved in memory. There, they help break down an embedded membrane protein called amyloid precursor protein into fragments, which group together in deposits found in people with Alzheimer’s disease.

Further study of gingipains and other microbial products could provide insight into a “critical early event in the initiation and progression of Alzheimer’s disease,” Ryder said.

Similarly, rheumatoid arthritis can be triggered by immune responses to other by-products of P. gingivalis, including protein antibodies that cause joint inflammation, according to Iain Chapple, BDS, PhD, professor of periodontology at the University of Birmingham [5].

Past research on links between the oral microbiome and systemic disease has even shown that these effects can travel a two-way street.

This is apparent in the research of Dana Graves, DDS, DMSc, professor of periodontics at University of Pennsylvania, whose work has examined the effects of diabetes on our microbiome, and vice versa [4].

“Diabetes impacts the mouth in a very profound way,” said Graves, adding that the inflammatory responses to bacteria caused by diabetes lead to disruption of the microbiome.

“This bidirectionality is something we saw first with diabetes, we’ve seen it now with rheumatoid arthritis, and it appears now that we’re starting to see it with chronic kidney disease,” Chapple said. “We need to start really digging down into [biological mechanisms] to understand more about that relationship.”

However, the verdict is still out on how much bacterial products such as gingipains contribute to disease. For Ryder and others, the existing data is insufficient — fully answering that question depends on carefully constructed clinical trials.

“When we’re trying to establish a link between something like the microbiome and the mouth and Alzheimer’s, association studies are important, the actual underlying biological mechanisms are important, but finally what sort of seals the deal, of course, is the actual effects of intervention,” Ryder said.

An ongoing clinical trial with more than 600 patients is evaluating the success of gingipain inhibitors in preventing symptoms of Alzheimer’s disease [10]. The results, expected by the end of this year, could carry implications not just for the treatment of Alzheimer’s disease but any disease with underlying roots in the oral microbiome.

Regardless of the results, it’s clear that breaking out the toothbrush and floss every day is crucial to our overall well-being.

 

References:

  1. Ursell LK, Metcalf JL, Parfrey LW, Knight R. 2012. Defining the human microbiome. Nutr Rev. 70 Suppl 1: S38-S44. https://doi.org/10.1111/j.1753-4887.2012.00493.x.
  2. Kumar P, D’Souza R, Shaddox L, Burne RA, Ebersole J, Graves D, Ryder MI, Chapple I. 2021. Killer Smile: The Link Between the Oral Microbiome and Systemic Disease [Conference presentation]. AAAS Annual Meeting [held virtually]. https://aaas.confex.com/aaas/2021/meetingapp.cgi/Session/27521.
  3. Deo PN, Deshmukh R. 2019. Oral microbiome: Unveiling the fundamentals. J Oral Maxillofac Pathol. 23(1): 122-128. doi:10.4103/jomfp.JOMFP_304_18.
  4. Graves DT, Ding Z, Yang Y. 2020. The impact of diabetes on periodontal diseases. Periodontol 2000. 82(1): 214-224. https://doi.org/10.1111/prd.12318.
  5. Lopez-Oliva I, Paropkari AD, Saraswat S, Serban S, Yonel Z, Sharma P, de Pablo P, Raza K, Filer A, Chapple I, et al. 2018. Dysbiotic subgingival microbial communities in periodontally healthy patients with rheumatoid arthritis. Arthritis Rheumatol. 70(7): 1008-1013. https://doi.org/10.1002/art.40485.
  6. Dioguardi M, Crincoli V, Laino L, Alovisi M, Sovereto D, Mastrangelo F, Lo Russo L, Lo Muzio L. 2020. The Role of Periodontitis and Periodontal Bacteria in the Onset and Progression of Alzheimer’s Disease: A Systematic Review. J Clin Med. 9(2): 495. https://doi.org/10.3390/jcm9020495.
  7. Arigbede AO, Babatope BO, Bamidele MK. 2012. Periodontitis and systemic diseases: A literature review. J Indian Soc Periodontol. 16(4): 487-491. https://doi.org/10.4103/0972-124X.106878.
  8. Curtis MA, Diaz PI, Van Dyke TE. 2020. The role of the microbiota in periodontal disease. Periodontol 2000. 83(1): 14-25. https://doi.org/10.1111/prd.12296.
  9. Ryder MI. 2020. Porphyromonas gingivalis and Alzheimer disease: Recent findings and potential therapies. J Periodontol. 91 Suppl 1: S45-S49. https://doi.org/10.1002/JPER.20-0104.
  10. Cortexyme Inc. 2021. GAIN Trial: Phase 2/3 Study of COR388 in Subjects With Alzheimer’s Disease. ClinicalTrials.Gov. Identifier NCT03823404. https://clinicaltrials.gov/ct2/show/NCT03823404.

It’s Not You, It’s Your Microbes: The Association Between Microbiota and Depressive Behavior in Mice

May 7, 2021

By Reshma Kolala, Medical & Molecular Microbiology ‘22

Author’s Note: A recent switch into the Microbiology major prompted me to explore recent developments in the field. I came across this study that examined the role of gut microbiota in brain function and mood regulation. With the globally rising prevalence of depression, this study provides some potential insight into the development of the disorder on a physiological level and provides a novel approach to anti-depression therapeutics. 

 

Afflicting nearly 350 million individuals annually, depression is a leading cause of disability worldwide. Despite the widespread effort to uncover the environmental and genetic basis of the disorder, the pathophysiology of depression remains elusive. This is attributed to the fact that, similar to other mental disorders, depression is the result of a complex interplay between several biological and societal factors [1]. Several studies have found that the pathology of depression is influenced by dysfunction in neuromodulatory systems, such as the endocannabinoid system (ECS). The ECS is composed of endocannabinoids (eCB), lipid-based neurotransmitters that regulate mood, emotions, and stress responses [2,3]. Another physiological factor that contributes to depression is the impairment of the hippocampal region of the brain, specifically hippocampal impaired neurogenesis, which contributes to depressive-like behaviors in rodents [4]. This is due to the fact that adult hippocampal neurogenesis has been shown to help mediate stress responses and depressive behavior. The dysfunction of these critical processes has recently been investigated in relation to symbiotic microbiota. 

It has been well established that the diversity of intestinal microbiota contributes to enhanced host function (particularly in immunity, metabolism, and the central nervous system) allowing an individual to better combat disease and regulate metabolic function [5,6,7]. Previous studies have demonstrated that dysbiosis, or altered intestinal microbial composition, has been found in depressed patients when compared to healthy controls [8].  It has also been observed that microbiota modulate anxiety symptoms in mice via the release of bacterial metabolites that may affect critical pathways in the brain [9]. Finally, colitis, a digestive disease characterized by inflammation in the colon, is influenced by gut microbiota and is commonly observed in depression patients [10]. Overall, these studies imply a potential association between intestinal microbial composition and depressive-like behaviors. The following study aims to examine the direct effect of gut microbiota on depressive behaviors in mice, allowing for a broader understanding of the physiological basis of depression and provide new avenues for therapeutics and potential treatment [11].

Researchers used unpredictable chronic mild stress (UCMS), a mouse model of stress-induced depression. To simulate stress, mice in the UCMS group were exposed to various stressors including cage tilting, altered cage bedding, foreign odor, and altered light/dark cycle. The mice in the UCMS group were exposed to two stressors a day for eight weeks. As expected, UCMS mice exhibited depressive-like behaviors such as decreased feeding and self-grooming behavior, consistent with apathetic behavior in those diagnosed with depression. UCMS mice also exhibited reduced hippocampal neurogenesis, confirming a previous study by Snyder et al. that noted this observation in rodents with depression [3].

Once depressive-like behaviors were established in UCMS mice, researchers conducted a fecal microbiota transplant (FMT) from mice exposed to stressors to mice that have not been exposed to any stressors. FMT’s are an innovative form of treatment in which a stool sample is collected from one individual and transplanted in the colon of another individual. This can be administered in various ways, such as a colonoscopy, oral capsules, or via a tube that stretches from the nose into the stomach or bowel [12]. In this study, mice received transplants via an oral gavage which involves the passage of a feeding needle down the esophagus. The purpose of an FMT is to populate the recipient intestine with diverse microorganisms that preferentially provide some benefit to the host. When the microbiota from the UCMS mice was transplanted into the healthy mice, the healthy mice exhibited decreased hippocampal neurogenesis and mimicked the depressive-like behaviors exhibited in the UCMS mice, although the healthy mice had not been exposed to any stressors. 

The effect of the FMT on recipient mice illustrated the influence of intestinal microbial composition on the host. Researchers in this study hypothesized that this was due to alterations in the host’s metabolism. To investigate this further, the concentration of multiple small molecule metabolites in bodily fluids was measured. This revealed a significant decrease in levels of several short-chain fatty acids which may have resulted from dysbiosis-induced changes. As fat is primarily broken down in the small intestine via chemical and mechanical processes, an altered microbial composition in the intestinal tract would unsurprisingly influence fat breakdown. More specifically, there was a decrease in the concentration of an eCB precursor, fatty acids containing arachidonic acid (AA), in recipient mice. As dysregulation of the ECS has been studied in association with depression, this finding in recipient mice aligns with the typical model of depression. To further understand the role of impaired eCB signaling in the recipient mice, researchers observed whether enhancing eCB signaling via dietary supplementation could alleviate the depressive-like behaviors observed in the recipient mice. It was found that recipient mice that were orally administered AA had reversed the depressive-like behaviors indeed by UCMS microbiota. Additionally, AA supplementation aided hippocampal neurogenesis.

To determine how UCMS microbiota affected the microbial composition of recipient mice, fecal microbiota from UCMS mice was sequenced using 16s rRNA. As 16s rRNA is present in all bacteria, the 16s rRNA gene is highly conserved and therefore, a useful tool to identify microbes within complex biological mixtures. The analysis revealed increased levels of Ruminoccacaae and Porphyromonodaceae and a decrease in Lactobacillacae. This finding supports previous studies that report an association between decreased Lactobacillacae and stress in mice. The differences in the microbial composition of recipient mice and donor UCMS mice were maintained eight weeks after transplantation. To test the influence of decreased Lactobacillacae in recipient mice, Lactobacillacae was orally administered similarly to AA supplantation. Dietary complementation of Lactobacillacae had a similar effect as AA supplantation, where depressive-like behaviors and impaired hippocampal neurogenesis were reversed.

Using mice, researchers discovered that the onset of depressive-like behaviors is triggered by a reduction in lipid metabolites. These lipid metabolites, more specifically endocannabinoids, bind to receptors in regions of the brain that control emotion and memory. Surprisingly, the concentrations of endocannabinoids are biochemically influenced by the gut microbiota. Although the mechanism by which this occurs has yet to be understood, these studies have elucidated the impact of gut microbiota beyond digestive function, revealing the extensive scope of microbial composition on healthy host function. This study specifically illustrates the importance of balanced gut microbiota for healthy neural and metabolic function and supports the potential use of dietary or probiotic supplementation as a treatment option for those diagnosed with depression. However, it is important to note that this area of research is relatively new and further studies are required to determine the translational capacity of studies related to the gut-brain axis from mice to humans. With consideration of the limitations of this study, this finding does still provide an intriguing avenue of treatment for mood disorders by introducing a novel physiological approach to mediate depressive-like symptoms. 

 

References

  1. Limbana, T., Khan, F., & Eskander, N. (2020). Gut Microbiome and Depression: How Microbes Affect the Way We Think. Cureus, 12(8). https://doi.org/10.7759/cureus.9966
  2. Hill, M. N., Hillard, C. J., Bambico, F. R., Patel, S., Gorzalka, B. B., & Gobbi, G. (2009). The therapeutic potential of the endocannabinoid system for the development of a novel class of antidepressants. Trends in pharmacological sciences, 30(9): 484–493. https://doi.org/10.1016/j.tips.2009.06.006
  3. Freitas, H. R., Ferreira, G., Trevenzoli, I. H., Oliveira, K. J., & de Melo Reis, R. A. (2017). Fatty Acids, Antioxidants and Physical Activity in Brain Aging. Nutrients, 9(11): 1263. https://doi.org/10.3390/nu9111263
  4. Snyder, J., Soumier, A., Brewer, M. et al. (2011) Adult hippocampal neurogenesis buffers stress responses and depressive behavior. Nature 476: 458–461. https://doi.org/10.1038/nature10287
  5. Belkaid, Y., & Hand, T. W. (2014). Role of the microbiota in immunity and inflammation. Cell, 157(1), 121–141. https://doi.org/10.1016/j.cell.2014.03.011
  6. Cani P. D. (2014). Metabolism in 2013: The gut microbiota manages host metabolism. Nature reviews. Endocrinology, 10(2): 74–76. https://doi.org/10.1038/nrendo.2013.240
  7. Sharon, G., Sampson, T. R., Geschwind, D. H., & Mazmanian, S. K. (2016). The Central Nervous System and the Gut Microbiome. Cell, 167(4): 915–932. https://doi.org/10.1016/j.cell.2016.10.027
  8. Jiang, H., Ling, Z., Zhang, Y., Mao, H., Ma, Z., Yin, Y., Wang, W., Tang, W., Tan, Z., Shi, J., Li, L., & Ruan, B. (2015). Altered fecal microbiota composition in patients with major depressive disorder. Brain, behavior, and immunity, 48: 186–194. https://doi.org/10.1016/j.bbi.2015.03.016
  9. Bercik, P., Denou, E., Collins, J., Jackson, W., Lu, J., Jury, J., Deng, Y., Blennerhassett, P., Macri, J., McCoy, K. D., Verdu, E. F., & Collins, S. M. (2011). The intestinal microbiota affect central levels of brain-derived neurotropic factor and behavior in mice. Gastroenterology, 141(2): 599–609. https://doi.org/10.1053/j.gastro.2011.04.052
  10. Kennedy, P. J., Clarke, G., Quigley, E. M., Groeger, J. A., Dinan, T. G., & Cryan, J. F. (2012). Gut memories: towards a cognitive neurobiology of irritable bowel syndrome. Neuroscience and biobehavioral reviews, 36(1): 310–340. https://doi.org/10.1016/j.neubiorev.2011.07.001
  11. Chevalier, G., Siopi, E., Guenin-Macé, L. et al. (2020). Effect of gut microbiota on depressive-like behaviors in mice is mediated by the endocannabinoid system. Nat Commun 11: 6363. https://doi.org/10.1038/s41467-020-19931-2
  12. Gupta, S., Allen-Vercoe, E., & Petrof, E. O. (2016). Fecal microbiota transplantation: in perspective. Therapeutic advances in gastroenterology, 9(2): 229–239. https://doi.org/10.1177/1756283X15607414

The Similarity of Human’s Microbiomes with Dogs

August 7, 2020

By Mangurleen Kaur, Biological Science, 23’

Author’s Note:  In one of my classes of basic biology, I got to learn about microbes. That class discussed some relationships between microbes and between human beings. One of the points that stuck in my mind was the relationship of microbes between humans and one of our favorite pets, dogs. By researching this topic, I found it so astounding that I decided to write about it. I hope this piece will be interesting not only for science-lovers but also for the general public.  

 

Both, inside and out, our bodies harbor a huge array of microorganisms. These microorganisms are a diverse group of generally minute life forms, which are called microbiota when they are found within a specific environment. Microbiota can refer to all the microorganisms found in an environment including bacteria, viruses, archaea, protozoa, and fungi. Furthermore, the collection of genomes from all the microorganisms found in a particular environment is referred to as a microbiome. According to the Human Microbiome Project (HMP), this plethora of microbes contribute more genes responsible for human survival than humans contribute. Researchers also estimated that the human microbiome consisted of 360 times more bacterial genes than human genes. Their results show that this contribution by microbes is critical for human survival. For instance, in the gastrointestinal tract bacterial genes are present, which allow humans to digest and absorb nutrients that otherwise would be unavailable. In addition to this, microbes also assist in the synthesis of many beneficial compounds, like vitamins and anti-inflammatory agents which our genome cannot produce. (4)

Where does this mini-ecosystem start from? The microbiome comes in our body as soon as we come out from the mother’s womb, we acquire them from the mother’s vagina and then, later on, by breastfeeding which plays a great role in making the microbes’ own unique community. There are several factors that influence the microbiome which include physiology, food, lifestyle, age, and environment. These are not only present in humans, but also in most animals and play a significant role in their health. For instance, gastrointestinal microorganisms exist in symbiotic associations with animals. Microorganisms in the gut assist in the digestion of feedstuffs, help protect the animal from infections, and some microbes even synthesize and provide essential nutrients to their animal host. This gives us an idea of how important these microorganisms are to our living system as a whole.(3)

Besides the human’s strong emotional connection with the dogs, there is also a biological relationship between the human and dog’s interactions. In context of this interesting relationship, research has been conducted. Computational biologist Luis Pedro Coelho and his colleagues at the European Molecular Biology Laboratory, in collaboration with Nestlé Research, studied the gut microbiome (the genetic material belonging to the microbiota) of beagles and retrievers. They found that the gene content of the dog’s microbiome showed more similarities to the human gut microbiome than to the microbiomes of pig or mice. When researchers mapped the gene content of the dog, mouse and pig microbiome in contrast to the human gut genes, they found that respectively 63%, 20% and 33% overlapped.(5) This shows the extensive similarities between human and dog’s gut microbiomes in comparison to other animals. Speaking on the discovery, Luis Pedro Coelho says: “We found many similarities between the gene content of the human and dog gut microbiome. The results of this comparison suggest that we are more similar to man’s best friend than we originally thought.” (1)

The University of Colorado Boulder did a study on the types of microbes present on the different parts of humans, to better understand the diversity and its significance for the human’s body. They conducted the study on 60 American families in which they sampled 159 people and 36 dogs. The team took samples from tongue, forehead, right and left palm and fecal samples to detect individual microbial communities. Through research, the researcher learned that people who own dogs are much more likely to share the same kinds of these “good” bacteria with their dogs. They have also learned that children who are raised with dogs are less likely than others to develop a range of immune-related disorders, including asthma and allergies. “One of the biggest surprises was that we could detect such a strong connection between their owners and pets,” said Knight, a faculty member at CU-Boulder’s BioFrontiers Institute.(6) The results found that adults who have a dog and they live together, share the greatest number of skin phylotypes while adults who neither have a dog nor live together share the least. 

The University of Arizona is also conducting another research study, with some other universities including UC San Diego, in which they are seeking healthy people from Arizona age 50 or older, who have not lived with dogs for at least the past 6 months. Then they are selecting persons who would like to live with the assigned dogs. The goal of the study is to see whether the dogs enhance the health of older people and work as probiotics (good bacteria). But this research is ongoing and the outcomes are not yet released. Rob Knight, Professor of Pediatrics and Computer Science & Engineering at UC San Diego and his lab studied microbiomes. Knight and his team found that the microbial community on adult skin are on average more similar to those of their own dogs than to others. They also found that cohabiting couples share more microbes with one another if they have a dog, compared with couples who don’t have a dog. Their research suggests that a dog’s owner can be identified just by analyzing the microbial diversity of the dog and its human, as they share microbiomes. These studies are finding a critical relationship that is very helpful in microbiology and the overall health field in science. (2) 

These studies reveal the various interesting relationships of microbiomes with us and other living beings. So far, the studies discussed how dog’s microbiomes are shared by the owner and how gene sequencing helps us to understand these connections. The growing understanding of this connection with microorganisms raises many other outstanding questions like what are the health benefits of a dog to a human? How can they help in preventing certain chronic diseases? This represents an exciting challenge for scientists and researchers to refine their understanding of microbiomes and find answers to these further emerging questions.

 

Work Cited

  1. “NIH Human Microbiome project defines normal bacterial makeup of the body”. National Institutes of Health, U.S. Department of Health and Human Services. www.nih.gov. Published on August 31, 2015. Acessed May 10, 2020
  2. Ganguly, Prabarna. “Microbes in us and their role in human health and disease”. www.Genome.gov. Published on May 29, 2019. Accessed May 10, 2020.
  3. “Dog microbiomes closer to humans’ than expected”. Research in Germany, Federal Ministry of Education and Research. www.researchingermany.org. Published on April 20, 2018. Accessed May 11, 2020.
  4. Trevino, Julissa. “A Surprising Way Dogs Are Similar to Humans.” www. Smithsonianmag.com. Published on April 23, 2018. Accessed February 11, 2020.
  5. Song, Se Jin, Christian Lauber, Elizabeth K Costello, Catherine A Lozupone, Gregory Humphrey, Donna Berg-Lyons, Gregory Caporaso, et al. “Cohabiting Family Members Share Microbiota with One Another and with Their Dogs.” eLife. eLife Sciences Publications, Ltd. elifesciences.org. Published on April 16, 2013. Accessed May 11, 2020. 
  6. Sriskantharajah, Srimathy. “Ever feel in your gut that you and your dog have more in common than you realized?” www.biomedcentral.com. Published on April 11, 2018. Accessed February 11, 2020.