1,721,049 research outputs found
Characterizing host protein post-translational modification in pathogenic Escherichia coli infection
No full textEnteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that causes significant morbidity and mortality worldwide. During infection, EPEC attaches to the intestinal epithelium and uses a molecular syringe-like nanomachine called a Type 3 Secretion system (T3SS) to secrete virulence ‘effector’ proteins directly into human cells, where they manipulate host cell signalling and mediate the characteristic features of infection, including intimate attachment to host cells, formation of actin-rich pedestals, disruption of the epithelial barrier, and dysregulated immune function and cell survival. Some effectors possess enzymatic activities that allow them to directly modify host proteins through post-translational modifications, including ubiquitination, phosphorylation, and proteolysis. However, the host targets of many effectors are unknown and their collective impact on global host post-translational modification has not been widely explored. This work utilizes proteomics tools to characterize the impact of EPEC infection and T3SS effectors on the host proteome. Here, ubiquitin proteomics were used to investigate the EPEC effector ubiquitin ligase NleG, exploring its impact on the host ubiquitome, and identifying and characterizing the relationship of NleG with a novel host substrate: Ataxin-3. We also developed a novel EPEC infection-proteomics workflow to characterize global changes in host protein post-translational modifications during important stages of infection. This method simultaneously profiled changes in protein abundance, ubiquitination, and phosphorylation due to EPEC infection, T3SS effector influences, and host response during early, mid-, and late infection. Infection-proteomics exhibited the highest coverage of the host proteome, ubiquitome, and phosphoproteome of any EPEC proteomics investigation to date, identifying thousands of EPEC infection-, exposure-, and T3SS-associated changes in proteins involved in cell processes regulated by EPEC during infection. These include innate immune signalling, actin remodelling, and cell survival pathways, while also identifying novel pathways targeted by EPEC. This work demonstrates the power of proteomics techniques to characterize impacts of EPEC infection on the host proteome by developing a novel approach to study how EPEC infection and effectors affect host post-translational modification and cell signalling throughout infection. Proteomics tools serve as valuable methods to study host-pathogen interactions and identifying molecular level changes underpinning characteristic phenotypes mediated by EPEC during infection.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat
Using population dynamics to uncover key barriers and signals involved in enteric infection
No full textThe gastrointestinal environment plays a critical role in shaping enteric infections. Host environmental factors create ‘bottlenecks’ which are restrictive events that can control the size and genetic diversity of invading bacterial populations. Despite this, little is known about the extent to which bacterial pathogens encounter bottlenecks, where bottlenecks occur, or how these events impact the pathogen founding population size (Nb′). I explored the population dynamics of murine gastrointestinal pathogen Citrobacter rodentium, a well-established model for human pathogenic Escherichia coli infection. To investigate bottlenecks, I created a library of 2,000+ isogenic but uniquely-tagged C. rodentium through the insertion of genomic barcodes. Following infection of mice, sequencing of recovered barcodes allowed for the quantification of population size. An average Nb′ of only 12-43 individual lineages was found across all timepoints and intestinal sites. We further identified passage through the stomach and intestinal escape to the systemic organs as major bottleneck events. These data demonstrate the effectiveness of bottlenecks in host protection, such that even highly adapted pathogens are subject to significant population loss. To fully appreciate their impact during infection, I manipulated key host protective bottlenecks caused by gastric acid and microbiota colonization. In both cases pathogen Nb′ increased (up to 6.8-fold and 35-fold following antacid treatment and treatment with the antibiotic vancomycin, respectively), as did host morbidity and mortality. Following host vancomycin treatment the differential abundance of growth-altering metabolites and commensal microbes which benefit pathogen colonization likely allows for lowered inter- and intra-species competition within the gut and promotes colonization by diverse pathogen lineages. Such environmental changes highlight the need for enteric pathogens to sense and respond to numerous environmental signals for successful intestinal navigation, demonstrating fluid regulation of bacterial processes even under typical infection conditions. Having established extreme pH as a barrier to infection, I further demonstrated that regional intestinal pH fluctuations are a signal of intestinal geography for invading pathogens, influencing C. rodentium growth, epithelial attachment and virulence. Altogether, the data presented in this thesis furthers our understanding of bottleneck events during enteric infection, demonstrating their potential to impact pathogen fitness and thereby the overall population-level diversity of epidemics.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat
Loss of Multicellular Behavior in Epidemic African Nontyphoidal Salmonella enterica Serovar Typhimurium ST313 Strain D23580
No full textNontyphoidal Salmonella enterica serovar Typhimurium is a frequent cause of bloodstream infections in children and HIV-infected adults in sub-Saharan Africa. Most isolates from African patients with bacteremia belong to a single sequence type, ST313, which is genetically distinct from gastroenteritis-associated ST19 strains, such as 14028s and SL1344. Some studies suggest that the rapid spread of ST313 across sub-Saharan Africa has been facilitated by anthroponotic (person-to-person) transmission, eliminating the need for Salmonella survival outside the host. While these studies have not ruled out zoonotic or other means of transmission, the anthroponotic hypothesis is supported by evidence of extensive genomic decay, a hallmark of host adaptation, in the sequenced ST313 strain D23580. We have identified and demonstrated 2 loss-of-function mutations in D23580, not present in the ST19 strain 14028s, that impair multicellular stress resistance associated with survival outside the host. These mutations result in inactivation of the KatE stationary-phase catalase that protects high-density bacterial communities from oxidative stress and the BcsG cellulose biosynthetic enzyme required for the RDAR (red, dry, and rough) colonial phenotype. However, we found that like 14028s, D23580 is able to elicit an acute inflammatory response and cause enteritis in mice and rhesus macaque monkeys. Collectively, these observations suggest that African S. Typhimurium ST313 strain D23580 is becoming adapted to an anthroponotic mode of transmission while retaining the ability to infect and cause enteritis in multiple host species.
IMPORTANCE: The last 3 decades have witnessed an epidemic of invasive nontyphoidal Salmonella infections in sub-Saharan Africa. Genomic analysis and clinical observations suggest that the Salmonella strains responsible for these infections are evolving to become more typhoid-like with regard to patterns of transmission and virulence. This study shows that a prototypical African nontyphoidal Salmonella strain has lost traits required for environmental stress resistance, consistent with an adaptation to a human-to-human mode of transmission. However, in contrast to predictions, the strain remains capable of causing acute inflammation in the mammalian intestine. This suggests that the systemic clinical presentation of invasive nontyphoidal Salmonella infections in Africa reflects the immune status of infected hosts rather than intrinsic differences in the virulence of African Salmonella strains. Our study provides important new insights into the evolution of host adaptation in bacterial pathogens
ZapE is a novel cell division protein interacting with FtsZ and modulating the Z-ring dynamics.
Full text linkBacterial cell division requires the formation of a mature divisome complex positioned at the midcell. The localization of the divisome complex is determined by the correct positioning, assembly, and constriction of the FtsZ ring (Z-ring). Z-ring constriction control remains poorly understood and (to some extent) controversial, probably due to the fact that this phenomenon is transient and controlled by numerous factors. Here, we characterize ZapE, a novel ATPase found in Gram-negative bacteria, which is required for growth under conditions of low oxygen, while loss of zapE results in temperature-dependent elongation of cell shape. We found that ZapE is recruited to the Z-ring during late stages of the cell division process and correlates with constriction of the Z-ring. Overexpression or inactivation of zapE leads to elongation of Escherichia coli and affects the dynamics of the Z-ring during division. In vitro, ZapE destabilizes FtsZ polymers in an ATP-dependent manner. IMPORTANCE Bacterial cell division has mainly been characterized in vitro. In this report, we could identify ZapE as a novel cell division protein which is not essential in vitro but is required during an infectious process. The bacterial cell division process relies on the assembly, positioning, and constriction of FtsZ ring (the so-called Z-ring). Among nonessential cell division proteins recently identified, ZapE is the first in which detection at the Z-ring correlates with its constriction. We demonstrate that ZapE abundance has to be tightly regulated to allow cell division to occur; absence or overexpression of ZapE leads to bacterial filamentation. As zapE is not essential, we speculate that additional Z-ring destabilizing proteins transiently recruited during late cell division process might be identified in the future
Diet and the microbiome in Parkinson's disease
No full textThe Mediterranean-Dietary Approaches to Stop Hypertension Intervention for Neurodegenerative Delay (MIND) diet has garnered significant attention in recent years due to its correlations with a reduced risk of Alzheimer’s disease (AD) and cognitive decline. However, its effects in Parkinson’s disease (PD) remain relatively unexplored. Dietary patterns strongly influence the microbiome, and its mechanisms of action may thus be partially attributable to microbial shifts. PD, a neurodegenerative disorder characterized by motor and nonmotor symptoms, exhibits a gut microbial phenotype that is relatively consistent across previous studies, although species-level analyses are lacking. My thesis aimed to determine whether the MIND diet represents a dietary pattern of interest with respect to PD etiology, characterize the species-level microbial patterns associated with PD, and determine whether the observed MIND-related microbial differences correlate with PD outcomes. Gut metagenomic sequencing was performed on a human cohort (PD=200, Ctrl=100) and diet intakes were assessed using food frequency questionnaires. Disease severity was assessed using levodopa equivalent dose (LED) and the Movement Disorders Society-Unified Parkinson’s Disease Rating Scale (MDS-UPDRS). Severity data was collected for 121 participants with PD over 5 years. MIND scores were strongly associated with later PD onset (P<0.006), especially in the female group, and weakly correlated with slower LED progression (Padj<0.08). Destabilization of the microbial network was observed in the PD group which corresponded to many taxonomic and functional differences, including a notable decrease in short-chain fatty acid producers and an increase in ribosomal genes. Random forest models suggested that these shifts were associated with faster disease progression. The MIND diet was primarily characterized by a reduction in pro-inflammatory serum C reactive protein (CRP) and an accompanying microbial pattern associated with lower CRP, both in PD and controls. People with PD whose microbiomes were categorized as significantly dysbiotic had lower MIND scores (P=0.004) and trended toward faster LED progression (P=0.13), though the latter association was underpowered and did not reach significance. Collectively, these findings support the MIND diet as a potential tool for reducing PD burden both pre- and post-PD diagnosis, and suggest that the underlying impact of the diet is related to the gut microbiome.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat
The role of breastmilk IgA in infant gut microbiota and immune development
No full textThe gut microbiota is highly dynamic in the first year of life and plays a pivotal role in immune development. Dysbiosis in the gut microbiota during this period has been linked to later development of numerous immune-mediated diseases, including allergies and asthma, but the specific host-microbe interactions that contribute to this phenomenon are still being uncovered. Secretory IgA (SIgA) is the most abundant antibody in the gut, and a key determinant of gut microbiota composition. During the first months of life, infants are not yet able to produce SIgA, receiving it instead through breastmilk. Breastmilk SIgA is known to protect infants against infections previously encountered by the mother. However, the effects of this antibody on infant gut microbiota composition, and thus immune development, have not been defined. This thesis investigates the role of breastmilk SIgA in guiding microbe-mediated immune imprinting in early life. Chapter 2 uses a mouse model of SIgA deficiency to show that maternal milk SIgA targets and limits the immunostimulatory microbe Segmented Filamentous Bacteria (SFB) in the neonatal murine gut, preventing premature activation of the intestinal Th17 cell responses and protecting against Th17-mediated asthma. Chapter 3 characterizes the human breastmilk SIgA repertoire in mothers enrolled in the CHILD Cohort Study, a longitudinal birth cohort in Canada, demonstrating a relationship between breastmilk SIgA-bacteria binding patterns and infant gut microbiota composition. This chapter identifies the bacterium Erysipelatoclostridium ramosum as an immunologically important target of breastmilk SIgA in the infant gut, and a potential human analogue to the well-studied and disease-relevant mouse pathobiont, SFB. Chapter 4 takes advantage of the techniques developed throughout Chapters 2 and 3 to elucidate the relationship between the maternal gut microbiota, breastmilk SIgA, and bacterial colonization dynamics in the offspring. This chapter confirms the ability of SIgA to mediate mother-to-offspring microbiota transfer. In summary, using in vivo models, human samples, longitudinal human health data, and cell culture, this work defines a significant role for breastmilk SIgA in guiding early-life gut microbiota and immune maturation, with important implications for future developments in preventative medicine and infant nutrition.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat
Hidden figures : an untold story of the impact of postnatal multiple micronutrient deficiencies on host phenome and gut microbiome
No full textWorldwide, approximately 340 million children, 50% of children < 5 years old, suffer from multiple micronutrient deficiencies. Termed “hidden hunger,” micronutrient deficiencies are silent contributors to death and disease burden. While vulnerability to micronutrient deficiencies remains throughout life, considerable evidence shows that early-life nutritional insults increase disease risk later in life through various mechanisms, including the gut microbiome, epigenetic modulation, and metabolic programming. This is expected, as the first 1000 days (first 2 years of life) is a critical period of host development and coincides with the assembly and maturation of the gut microbiome. The global malnutrition literature lacks relevant biological models to investigate the impact of early life micronutrient deficiencies and methods to study the long-term effects. To address this gap, we created a novel multiple micronutrient deficient murine model to characterize the host phenome, set of phenotypes expressed by an organism, tissue, cell, or organ, and the gut microbiome. I further developed this model to investigate the long-term effects of postnatal micronutrient deficiency exposure in a condition known as the double burden of malnutrition. We used weanling C57Bl/6N mice fed an experimental control or a multiple low-micronutrient diet deficient in (vitamin A, B12, B9, zinc, and iron) for 4-weeks. In the second experiment, at the end of 4-weeks, mice were challenged with a high-fat diet for 12 weeks. Characterization of the host phenome within growth, metabolism, and methylome domains revealed significant stunting, altered body composition, and lipid profile and dysregulated glucose metabolism. Metagenomics sequencing of the microbiome showed significant dysbiosis driven by an expansion of relative and absolute (10¹⁰ CFU) abundance of Enterobacteriaceae, namely Klebsiella and Enterobacter. Despite the lack of antibiotic exposure, we observed significant expansion in antibiotic resistance mechanisms, namely efflux pumps, degradation enzymes, permeability, target site, and antibiotic resistance genes at 4-weeks. Moreover, postnatally micronutrient deficient mice later exposed to a high-fat diet had greater glucose intolerance and insulin resistance than control mice fed a high-fat diet. These findings highlight several unappreciated roles of postnatal multiple micronutrient deficiencies in the global threat of antibiotic resistance dissemination and the risk of non-communicable disease in low-to-mid-income countries.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat
Exploring bacterial communication signaling in the context of the intestinal microbiota
No full textThe full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat
The role of gut microbiota in Parkinson's disease
No full textParkinson’s disease (PD) is a neurodegenerative disorder characterized by motor
dysfunction. Non-motor symptoms including gastrointestinal (GI) dysfunction and mood
disorders (such as depression) are also particularly common. GI symptoms include constipation,
and PD patients display altered gut microbiota composition. Evidence in animal models points
towards a potential causal role for the microbiota in mediating PD pathology. However, the
constipation, medication use, and lifestyle habits of PD patients can also be expected to change
microbiota composition. In this thesis, I explore the role of the gut microbiota in PD, in terms of
the degree that it is shaped by the disease state and its ability to mediate disease symptoms.
Using a transgenic mouse model of PD that displays motor deficits, GI dysfunction, and
behavioral alterations, I assessed how broad alterations to the gut microbiota impacted the motor
and non-motor phenotype. I found that both depletion of the microbiota through antibiotics, and
a shift towards a healthy wild-type mouse microbiota, had a minimal impact on the PD-like
symptoms. This suggested that the PD-like transgenic state of this model may drive the disease
phenotype to a greater extent than the microbiota. Similarly, I demonstrated that the decreased
abundance of Lachnospiraceae and decreased abundance of Ruminococcaceae and Oscillospira
observed in PD patients may be a result of constipation by treating PD mice with laxatives that
reversed these shifts. Lachnospiraceae abundance was also found to be decreased by treatment of this model with the PD medications L-DOPA and carbidopa.
Conversely, different antibiotic treatment regimens were able to shift the microbial
community and alter GI transit time in PD mice. Specific bacterial taxa, such as Lachnospiraceae
(Ruminococcus), were associated with transit time – indicating a potential to treat PD
constipation via the microbiota. Furthermore, treatment of PD mice with PD medications had a beneficial effect on constipation and depression-like behavior, potentially through increasing the
abundance of Turicibacter and promoting butyrate production.
This thesis demonstrates that certain PD-associated microbiota alterations may be a result
of slowed GI transit or the presence of medications. However, specific shifts to the gut
microbiota may in turn mediate non-motor symptoms in PD.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat
Multi-omics investigation of host-pathogen interactions during pathogenic Escherichia coli infection
No full textInfectious diarrheal diseases are the third leading cause of mortality in young children, many of which are driven by Gram-negative bacterial pathogens, including enteropathogenic Escherichia coli (EPEC). This thesis employed an amalgamation of omics, in silico prediction, and conventional molecular biology tools to investigate the host-pathogen interactions during EPEC infection, primarily focusing on the systems-level and protein interactions. We employed a dual RNA-sequencing approach to investigate the host and microbe physiology during EPEC infection of an intestinal epithelial cell line (Caco-2/TC-7) with a focus on the role of the Type III Secretion System (T3SS). Our findings showed that T3SS was used by EPEC to suppress various host responses, including immune signaling and apoptosis. We hypothesized that microRNAs (miRNAs) were important for mediating host and pathogen behaviors and identified differentially expressed miRNAs that played a role in suppressing cell death and altering cytokine secretion in response to infection. With regards to bacterial transcriptome, we observed drastic upregulation of the Type II Secretion System (T2SS) genes in EPEC upon host cell contact. To explore the role of the T2SS during natural host infection, we used Citrobacter rodentium, a murine enteric pathogen, as a model of EPEC-caused disease. We demonstrated that this system was functional in vitro with potential roles in intestinal mucin degradation. During host infection, loss of the T2SS or predicted effectors led to a significant colonization defect and lack of systemic spread. Finally, apoptosis was among the pathways with a T3SS-associated pattern of dysregulation during infection. This prompted us to investigate the function of a pro-apoptotic T3SS effector, Map, due to the lack of understanding of its mechanism of action. Through a combination of proximity labelling mass spectrometry and deep learning interaction prediction algorithm, we identified several Map partners, several of which were involved in the electron transport chain (ETC). Collectively, this thesis provides the first survey of the host and bacterial transcriptomes during EPEC infection, the first confirmation of the importance of the C. rodentium T2SS for robust infection in vivo, and identification of novel host mitochondrial proteins interacting with EPEC effector Map.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat
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