Overexpression of tlr5 affects gut microbiota resistance to Salmonella infection

doi:10.21203/rs.3.rs-4517269/v1

Download PDF

Research Article

Overexpression of tlr5 affects gut microbiota resistance to Salmonella infection

https://doi.org/10.21203/rs.3.rs-4517269/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Background

The gut microbiome and the host immune system interact synergistically to maintain intestinal health and defend against infections. Toll-like receptor 5 (TLR5), known for recognizing bacterial flagellin, plays a pivotal role in this regulatory network. Despite significant advances in understanding the gut microbiome and TLR5, the precise role of TLR5 in gut health and infection resistance remains unclear. This study aimed to elucidate the role of TLR5 in regulating gut microbiota and metabolites and their impact on resistance to Salmonella infection using a TLR5 intestinal-specific overexpression mouse model.

Methods

Fecal microbiota and metabolite transplantation experiments were conducted using feces from TLR5-overexpressing mice (TLR5+/+ group) and wild-type mice (WT group) into C57 mice, creating four experimental groups: Flora-TLR5-C57, Metabolite-TLR5-C57, Flora-WT-C57, and Metabolite-WT-C57. Mice were infected with Salmonella, and various parameters including survival time, fecal Salmonella load, and alterations in gut microbiota and metabolites were assessed.

Results

Despite notable shifts in gut microbiota composition and metabolite profiles induced by TLR5 overexpression, no significant differences in fecal Salmonella load or survival time were observed across the experimental groups. TLR5-overexpressing mice exhibited an enrichment of beneficial bacterial taxa and significant changes in metabolites; however, these alterations did not correlate with enhanced resistance to Salmonella infection.

Conclusion

Our findings suggest that while TLR5 overexpression significantly modifies gut microbiota and metabolite profiles, these changes do not directly enhance resistance to Salmonella infection. The immune response to Salmonella is multifaceted and involves various components beyond the gut microbiota and metabolites. Further research is required to elucidate the detailed molecular pathways through which TLR5 influences infection outcomes and to explore potential combined therapeutic strategies for enhancing host defenses against enteric pathogens.

TLR5

Gut Microbiota

Salmonella Infection

Immune Response

Intestinal Health

The synergy between the gut microbiome and the host immune system is crucial in maintaining intestinal health and defending against infections. The Toll-like receptor (TLR) family, especially TLR5, plays a pivotal role in this complex regulatory network. TLR5, known for recognizing bacterial flagellin, is integral to modulating immune responses and maintaining gut microbial balance^1,2.

Despite the significant advances in our understanding of the gut microbiome and TLR5, several critical questions remain unanswered. Primarily, the precise role of TLR5 in gut health, particularly its function in infection resistance, needs further elucidation³. Additionally, while overexpression of TLR5 has been suggested to enhance immune responses, the detailed molecular mechanisms underpinning this regulation are not fully understood⁴.

This study aimed to bridge these knowledge gaps by utilizing a TLR5 intestinal-specific overexpression mouse model to explore the cooperative regulatory mechanisms between intestinal microorganisms and resistance to Salmonella infection. We conducted fecal microbiota and metabolite transplantation experiments to investigate whether changes in gut microbiota and metabolite profiles due to TLR5 overexpression could enhance infection resistance. Contrary to our expectations, these transplantation experiments did not yield positive results, indicating that TLR5-induced alterations in the gut microbiome and metabolites do not directly confer enhanced resistance to Salmonella infection.

In light of these findings, this article seeks to explore the intricate relationships between TLR5, the gut microbiome, and infection resistance. By delving into the molecular pathways regulated by TLR5 and its interactions with gut microorganisms, we aim to provide new insights into the mechanisms of immune regulation and infection defense. A deeper understanding of these processes will inform the development of innovative strategies to maintain intestinal health and combat microbial infections^5,6.

Downregulation of Colonic TLR5 Receptor Content in Mice upon Salmonella Infection

In our study, we focused on the changes in Toll-like receptor 5 (TLR5) receptor content in the colons of mice on the fifth day following Salmonella infection. TLR5 plays a crucial role in the host's immune system, responsible for recognizing bacterial flagellin and initiating immune responses⁶. On the fifth day following Salmonella infection, we observed a significant downregulation of TLR5 receptor content in the colon tissue of mice. These results clearly indicate the suppression of TLR5 during the early stages of Salmonella infection. Surprisingly, the flow cytometry and immunofluorescence experiments, which primarily focus on assessing the cell surface TLR5 protein content, did not reveal significant differences in TLR5 expression within colonic tissue before and after Salmonella infection. These results suggest that the impact of Salmonella infection may be more prominent on the intracellular processes involved in TLR5 protein generation within the cells rather than affecting the pre-existing TLR5 content on the cell surface⁷. The complexity of TLR5 regulation in response to infection becomes evident, highlighting the need for further investigations to elucidate the specific cellular mechanisms involved.

On the fifth day after Salmonella infection, the mRNA levels of TLR5 were determined by qRT-PCR and the protein levels were examined by western blot.

Impact of TLR5 Overexpression on Salmonella Infection Outcome

In this study, we aimed to investigate the impact of TLR5 downregulation on the prognosis of Salmonella-infected mice. To achieve this, we generated transgenic mice with intestinal overexpression of TLR5, denoted as TLR5+/+. These TLR5-overexpressing mice, along with a control group of wild-type (WT) mice, were subjected to Salmonella infection at the specified dosage. The results revealed several significant positive outcomes in the TLR5+/+ mice compared to WT mice following Salmonella infection: they exhibited a significantly extended survival time, displayed a lower Salmonella content in their feces, and notably, had a longer colon length on the fifth day post-infection. Shortening of the colon is considered to be one of the manifestations of chronic damage to the colon⁸. However, despite these positive findings, no substantial disparities in spleen weight were observed between the two groups on the fifth day post-infection, and pathological damage to the colon tissues showed no significant differences post-infection⁹, suggesting comparable inflammatory responses. These results underscore the complex role of TLR5 in the host response to Salmonella and its potential as a target for interventions aimed at enhancing host defense against this pathogen, while further investigations are needed to elucidate the underlying mechanisms driving these observed effects.

(A)Kaplan-Meier survival curve. (B)Salmonella in faeces on day 5 after infection(CFU/g). (C) mRNA levels of TLR5 in TLR5 intestine-specific overexpressing mice (TC group) and their control mice (WC group). (D) Colon, liver, and spleen tissues from control mice prior to infection (WC) and five days post Salmonella infection (WS), alongside corresponding tissues from TLR5+/+ mice before (TC) and after (TS) infection. From left to right, the panels display the tissue samples from the various mouse groups. (E) Spleen weight before and after infection across different mouse groups. (F) Colon length before and after infection across different mouse groups. (G) Colon pathology in WT mice and TLR5+/+ mice following Salmonella infection.

Exploring the Gut Microbiota Diversity through 16S Sequencing Analysis

In order to study the specific mechanism by which the intestinal microenvironment affects the reproduction of Salmonella, we examined the intestinal components of mice in the TLR5 overexpression group and the control group. The 16s sequencing results and non-targeted metabolite sequencing results showed that the intestinal flora composition and metabolite composition of mice in the TLR5 and WT groups were different.The linear discriminant analysis showed that Bacteroides, Akkermansia, Burkholderiales, Ruminococcus were significantly enriched in the WT mice, whereas the Muribaculaceae, Rikenella, Lachnospiraceae were significantly enriched in the TLR5 group.

TLR5 overexpression alters the gut microbiota of mice. (A): Fecal microbiota composition of TLR5+/+ and WT mice at the genus level.(B): Principal component analysis (PCoA) of faecal microbiota in TLR5+/+ and WT mice. The significance of two separated clusters was measured with the Adonis test. (C). LEfSe analysis showing bacterial taxa that were significantly different in abundance between TLR5+/+ and WT. (D).Differential genes in genus classification. (E). PICRUSt analysis was performed for functional profile predictions of microbiota based on 16S rRNA marker gene.

Non-targeted Metabolomics Reveals Differential Fecal Metabolite Profiles in TLR5 Overexpressing Mice

In this study, we conducted a comprehensive metabolomic analysis¹⁰ to explore potential differences in fecal metabolite compositions between Toll-like receptor 5 overexpressing mice (TLR5+/+ group) and control mice (WT group). We employed a range of analytical approaches, including multivariate statistical analyses¹¹, rank-sum tests, differential metabolite identification, metabolite clustering, and pathway enrichment analysis.Firstly, we employed multivariate statistical analyses, including OPLS-DA¹², to model and analyze the fecal metabolomic data of both mouse groups. To confirm the statistical significance of observed differences, rank-sum tests were conducted. Results demonstrated significant differences in the fecal metabolite profiles between the two groups, in both anionic and cationic modes¹³.Subsequently, differential metabolite identification was employed to identify metabolites that exhibited significant increases or decreases in abundance between the TLR5+/+ and WT groups. This facilitated the rapid identification of key metabolites potentially associated with TLR5 overexpression. Our findings revealed that 3beta-3-Hydroxy-18-lupen-21-one¹⁴, Ethyl (E)-7,9-decadienoate, and Methylpyrazine¹⁵ were significantly elevated in the TLR5+/+ group, while Naringenin and Gluconolactone were more abundant in the WT group.Metabolite clustering analysis grouped similar metabolites together, unveiling associations within metabolic pathways and biological processes. This step aided in understanding the interrelationships among metabolites and their collaborative roles in specific biological processes.To gain deeper insights into the biological significance of differential metabolites, we conducted KEGG pathway enrichment analysis, revealing significant enrichment of differential metabolites in pathways such as Biosynthesis of other secondary metabolites, Lipid metabolism, and Chemical structure transformation maps.

Finally, as depicted in the figures, we performed correlation analysis between metabolites and microbial composition, shedding light on potential connections between the gut metabolome and microbiota.

(A).OPLS-DA models were carried out by comparison of two groups. (B). Volcano plot depicting differential fecal metabolites between TLR5-overexpressing (TLR5+/+) and control (WT) mice, highlighting metabolites with statistical significance. (C). Cluster heatmap illustrating the hierarchical clustering of differential fecal metabolites, revealing distinct metabolic profiles between TLR5-overexpressing (TLR5+/+) and control (WT) mice. (D). Variable Importance in Projection (VIP) plot showcasing the significance of differential fecal metabolites in distinguishing between TLR5-overexpressing (TLR5+/+) and control (WT) mice. E. KEGG Pathway Enrichment Analysis of Differential Fecal Metabolites: Representation of the enriched KEGG metabolic pathways linked to the identified differential fecal metabolites, shedding light on their potential functional roles in specific biological processes.

Effects of Fecal Microbiota Transplantation from TLR5 Overexpressing Mice on Salmonella Load and Survival in C57 Mice

In this study, fecal microbiota transplantation experiments were conducted by separating bacterial flora and metabolites from the feces of TLR5-overexpressing mice (TLR5+/+ group) and wild-type mice (WT group). The separated components were then transplanted into C57 mice, resulting in four distinct experimental groups: Flora-TLR5-C57 Mice, Metabolite-TLR5-C57 Mice, Flora-WT-C57 Mice, and Metabolite-WT-C57 Mice. Each group was subsequently infected with Salmonella. Despite the distinct gut microbiota and intestinal metabolite profiles induced by TLR5 overexpression in colon epithelial cells compared to the control group, no significant differences in Salmonella load were observed in the feces across all four groups. Additionally, survival times did not differ between the groups following Salmonella infection. According to prior literature, the gut microbiota is expected to confer some level of resistance to Salmonella infection¹⁶; however, the results suggest that the transplantation of specific bacterial flora or metabolites does not directly enhance resistance to Salmonella infection.It is possible that certain bacteria may become inactive quickly once removed from the mouse gut environment, which necessitates further research¹⁷.

(A). Fecal Salmonella load in different groups of C57 mice after fecal microbiota or metabolite transplantation and Salmonella infection.The heatmap shows the fecal Salmonella load in C57 mice that received either bacterial flora or metabolite transplants from TLR5-overexpressing mice or wild-type mice. Four groups were analyzed: Metabolite-TLR5-C57, Metabolite-WT-C57, Flora-TLR5-C57, and Flora-WT-C57. The Salmonella load was measured at three different time points: Day 1 (D1), Day 3 (D3), and Day 5 (D5) post-Salmonella infection. The color scale represents the Salmonella load, with yellow indicating the highest load and purple the lowest. No significant differences in Salmonella load were observed between any of the groups at any of the time points. (B) Survival analysis of C57 mice following fecal microbiota or metabolite transplantation and subsequent Salmonella infection.

Mice

In this study, two mouse strains were employed: C57BL/6 mice, obtained from the School of Pharmacy at Fudan University, and TLR5 Intestine-Specific Overexpression Mice (TLR5+/+), genetically modified to overexpress TLR5 specifically in the intestine and provided by Shanghai Southern Model Organisms, Inc. A control group of mice with a pseudo-gene-editing background (wild type, WT) was also included for comparative purposes. These distinct mouse strains allowed for the investigation of TLR5 effects within the context of the study, offering valuable insights into host responses and TLR5's role in the experimental outcomes.

Euthanasia Methods:

The animals used in this study were anesthetized using 10% chloral hydrate administered via intraperitoneal injection. The specific dosage used was 4 ml/kg, determined based on previous studies to ensure effective anesthesia. After the animals were fully anesthetized and unconscious, euthanasia was performed using cervical dislocation.

Salmonella

In this study, mice were housed under standard laboratory conditions and euthanized on the fifth day post-infection. The mice were randomly assigned to either the Infection Group or the Healthy Control Group. In the Infection Group, mice were orally infected with Salmonella enterica at a dose of 10^7 Colony-Forming Units (CFU) via gastric gavage, serving as the experimental group to assess the effects of Salmonella infection. The Healthy Control Group received an equivalent volume of sterile physiological saline via gastric gavage as a control. This model allowed for the evaluation of host responses to Salmonella infection and the impact of TLR5 overexpression, ensuring ethical treatment throughout the study.

Tissue collection and RNA extraction

On the fifth day postinfection, mouse colon tissue was immediately removed and snap frozen in liquid nitrogen. Total RNA was extracted from colon tissue using the Novizan Total RNA extraction kit (Invitrogen), following the manufacturer's instructions.

Real-time quantitative polymerase chain reaction (qPCR) analysis

Starting from total RNA, cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Invitrogen). Novizan PowerUp SYBR Green Master Mix (Invitrogen) was used to perform qPCR analysis to quantitatively determine the expression level of TLR5 gene. Normalization was performed using GAPDH as an internal reference gene. Each sample was subjected to three qPCR experiments with technical replicates, using the ABI StepOnePlus real-time PCR system.

Western blot analysis

Another part of the colon tissue was subjected to protein extraction in RIPA buffer adding Novizan Halt protease inhibitor and phosphatase inhibitor. Proteins were separated using the Bio-Rad Rapid Gel Kit (Bio-Rad) and subsequently transferred to polyacrylamide gels by wet electrophoresis. Western blotting was performed using a Novizan TLR5-specific antibody and β-actin protein was used as an internal reference protein for normalization.

Gut Microbiota Transfer

In this study, we conducted fecal microbiota transplantation (FMT) and metabolite transplantation experiments to investigate the impact of transferred gut microbiota and metabolites on the survival time and fecal Salmonella content of recipient mice. Specifically, fecal samples were collected from two distinct donor groups: TLR5+/+ mice (TLR5 group) and wild-type (WT) mice. These samples were centrifuged twice to separate the bacterial flora and metabolites. The separated components were then transplanted into recipient C57 mice for a continuous duration of 14 days, resulting in the establishment of four recipient groups:Flora-TLR5-C57 Mice: C57 mice that received bacterial flora from TLR5-overexpressing mice.Metabolite-TLR5-C57 Mice: C57 mice that received metabolites from TLR5-overexpressing mice.Flora-WT-C57 Mice: C57 mice that received bacterial flora from wild-type mice.Metabolite-WT-C57 Mice: C57 mice that received metabolites from wild-type mice.Following the transplantation period, all four groups were subjected to Salmonella infection. Subsequently, we monitored and recorded the survival duration of the mice as well as the Salmonella content in their feces at multiple time points.

Intestinal content collection and DNA extraction

On day five post-infection, mice were humanely euthanized and intestinal contents were harvested. The DNA extraction kit provided by Shanghai Meiji Biological Co., Ltd. was used to extract total DNA from intestinal contents according to its instructions.

16S rRNA gene sequencing analysis

PCR amplification of the V3-V4 region of the 16S rRNA gene was performed on the DNA samples, using primers provided by Shanghai Meiji Biological Company, and constructing the library according to its recommendations. Use the Illumina MiSeq platform for high-throughput sequencing to obtain raw sequencing data. Data processing includes denoising, removing redundancy, merging, and generating OTUs. For further analysis, the bioinformatics tools and analysis procedures recommended by Shanghai Meiji Biotechnology Co., Ltd. were used.

Non-target metabolome sequencing analysis

Non-target metabolome sequencing of intestinal content DNA was performed on the Illumina HiSeq platform to obtain raw sequencing data. Data analysis included quality control, alignment to reference genome, gene quantification and differential expression analysis, and was performed in accordance with the recommendations of Shanghai Meiji Biotechnology Company.

Data analysis

Relative quantification of gene expression was calculated using the ΔΔCt method. ImageJ software was used to analyze the intensity of protein bands in Western blots. The results are expressed as mean ± standard deviation. Appropriate methods were used for statistical analysis. P values less than 0.05 were considered statistically significant.

This study aimed to investigate the role of TLR5 overexpression in modulating gut microbiota composition and metabolite profiles, as well as its impact on resistance to Salmonella infection. Despite significant changes in the gut microbiota and metabolite profiles induced by TLR5 overexpression, our results indicate that these alterations do not enhance resistance to Salmonella infection.

TLR5 overexpression led to notable shifts in gut microbiota composition, particularly an enrichment of beneficial bacterial taxa such as Muribaculaceae and Lachnospiraceae. These taxa are often associated with improved gut health and enhanced immune responses. However, despite these favorable changes in microbial composition, there was no significant reduction in fecal Salmonella load, suggesting that TLR5-induced changes in the gut microbiota are not sufficient to confer enhanced resistance to infection.

Metabolomic analysis revealed significant differences in the fecal metabolite profiles between TLR5-overexpressing mice and wild-type controls. TLR5-overexpressing mice exhibited higher levels of metabolites such as 3beta-3-Hydroxy-18-lupen-21-one, Ethyl (E)-79-decadienoate, and Methylpyrazine. In contrast, metabolites like Naringenin and Gluconolactone were more abundant in the wild-type group. Despite these differences, the altered metabolite profiles did not correlate with improved infection outcomes, indicating that these metabolic changes do not directly contribute to enhanced Salmonella resistance .

Survival analysis and fecal Salmonella load measurements further supported these findings. Both Flora-TLR5-C57 and Metabolite-TLR5-C57 groups exhibited similar survival rates and Salmonella loads as their wild-type counterparts. These results suggest that while TLR5 can modulate gut microbiota and metabolites, these changes do not translate into measurable benefits in terms of infection resistance.

One possible explanation for the lack of enhanced resistance is that the immune response to Salmonella infection is multifaceted and involves various components of the immune system beyond the gut microbiota and metabolites¹⁸. TLR5’s role in recognizing bacterial flagellin and initiating immune responses might be overshadowed by the contributions of other Toll-like receptors and immune pathways that are equally, if not more, critical in managing Salmonella infections. Additionally, the specific interactions between TLR5-induced microbial changes and the host immune system may not be robust enough to significantly impact pathogen clearance.

These findings highlight the complexity of host-pathogen interactions and the need for a deeper understanding of the mechanisms by which TLR5 modulates immune responses and microbial interactions. Future research should focus on elucidating the detailed molecular pathways through which TLR5 influences infection outcomes. Investigating the interactions between TLR5 and other TLRs, as well as their combined effects on gut microbiota, may provide deeper insights into the regulatory networks governing gut immunity .

Furthermore, exploring the potential of combining TLR5 modulation with other therapeutic strategies could open new avenues for enhancing host defenses against enteric pathogens. For instance, using probiotics or prebiotics to selectively promote the growth of beneficial microbes in conjunction with TLR5 activation might enhance overall immune responses and improve resistance to infections .

In conclusion, this study demonstrates that while TLR5 overexpression significantly alters gut microbiota and metabolite profiles, these changes do not enhance resistance to Salmonella infection. These findings underscore the complexity of microbial-host interactions and the need for a comprehensive understanding of the mechanisms underlying gut immunity and infection resistance. Further studies are essential to uncover the specific pathways and interactions that can be targeted to improve host defense against enteric pathogens.

Ethics statement

The animal study was reviewed and approved by the Animal Ethics Committee of the School of Pharmacy, Fudan University. In this study, two mouse strains were employed: C57BL/6 mice, obtained from the School of Pharmacy at Fudan University, and TLR5 Intestine-Specific Overexpression Mice (TLR5+/+), genetically modified to overexpress TLR5 specifically in the intestine and provided by Shanghai Southern Model Organisms, Inc. A control group of mice with a pseudo-gene-editing background (wild type, WT) was also included for comparative purposes. I have obtained full consent from the providers of these animals for their use in this study.

Acknowledgments

Not applicable

Consent for publication

Not applicable

Funding

The study was supported by the Shanghai Minhang District Health Committee: Application of Bedside Cardiopulmonary Combined with Ultrasound in the Evaluation of Severe Pneumonia (2019MW11), and the Institute Level Project of Minhang Hospital, Fudan University: Effect of EpCAM Targeting Recombinant SVV-IL-12 Nanodrug Delivery System on Neuroendocrine Tumor (2019MHJC09).

Authors’ contributions

Xiang Li and Xu Zhao designed the study. Chen Lei and Yue Wang carried out experiments and wrote the manuscipt, Lijing Jiang and Lu Zhang revised the paper, Weiyi Tang collected mice specimens and related information. Zhi Li contributed to analysing the data. All authors reviewed the results and approved the final version of the manuscript.

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Yassour, M. et al. Natural history of the infant gut microbiome and impact of antibiotic treatment on bacterial strain diversity and stability. Science Translational Medicine8, 343ra381 (2016). https://doi.org/10.1126/scitranslmed.aad0917
Hug, H., Mohajeri, M. H. & La Fata, G. Toll-Like Receptors: Regulators of the Immune Response in the Human Gut. Nutrients10 (2018). https://doi.org/10.3390/nu10020203
Zheng, W. et al. Toll-like receptor-mediated innate immunity against herpesviridae infection: a current perspective on viral infection signaling pathways. Virol J17, 192 (2020). https://doi.org/10.1186/s12985-020-01463-2
Oh, J. Z. et al. TLR5-mediated sensing of gut microbiota is necessary for antibody responses to seasonal influenza vaccination. Immunity41, 478-492 (2014). https://doi.org/10.1016/j.immuni.2014.08.009
Menon, B. B., Kaiser-Marko, C., Spurr-Michaud, S., Tisdale, A. S. & Gipson, I. K. Suppression of Toll-like receptor-mediated innate immune responses at the ocular surface by the membrane-associated mucins MUC1 and MUC16. Mucosal Immunol8, 1000-1008 (2015). https://doi.org/10.1038/mi.2014.127
Li, W. et al. Activation of NLRC4 downregulates TLR5-mediated antibody immune responses against flagellin. Cell Mol Immunol13, 514-523 (2016). https://doi.org/10.1038/cmi.2015.33
Van Maele, L. et al. Airway structural cells regulate TLR5-mediated mucosal adjuvant activity. Mucosal Immunol7, 489-500 (2014). https://doi.org/10.1038/mi.2013.66
Lee, K.-E. et al. The extracellular vesicle of gut microbial Paenalcaligenes hominis is a risk factor for vagus nerve-mediated cognitive impairment. Microbiome8, 107 (2020). https://doi.org/10.1186/s40168-020-00881-2
Beristain-Covarrubias, N. et al. Salmonella-induced thrombi in mice develop asynchronously in the spleen and liver and are not effective bacterial traps. Blood133, 600-604 (2019). https://doi.org/10.1182/blood-2018-08-867267
Hamzeiy, H. & Cox, J. What computational non-targeted mass spectrometry-based metabolomics can gain from shotgun proteomics. Curr Opin Biotechnol43, 141-146 (2017). https://doi.org/10.1016/j.copbio.2016.11.014
Hidalgo, B. & Goodman, M. Multivariate or multivariable regression? Am J Public Health103, 39-40 (2013). https://doi.org/10.2105/AJPH.2012.300897
Lee, L. C., Liong, C.-Y. & Jemain, A. A. Partial least squares-discriminant analysis (PLS-DA) for classification of high-dimensional (HD) data: a review of contemporary practice strategies and knowledge gaps. Analyst143, 3526-3539 (2018). https://doi.org/10.1039/c8an00599k
Lachin, J. M. Nonparametric Statistical Analysis. JAMA323, 2080-2081 (2020). https://doi.org/10.1001/jama.2020.5874
Kumari, S., Saini, R., Bhatnagar, A. & Mishra, A. HR-LCMS and evaluation of anti-diabetic activity of Hemidesmus indicus (anantmool): Kinetic study, and molecular modelling approach. Comput Biol Chem105, 107896 (2023). https://doi.org/10.1016/j.compbiolchem.2023.107896
Eng, T. et al. Production of tetra-methylpyrazine using engineered Corynebacterium glutamicum. Metab Eng Commun10, e00115 (2020). https://doi.org/10.1016/j.mec.2019.e00115
Rogers, A. W. L., Tsolis, R. M. & Bäumler, A. J. salmonella versus the Microbiome. Microbiol Mol Biol Rev85 (2021). https://doi.org/10.1128/MMBR.00027-19
Porcari, S. et al. Key determinants of success in fecal microbiota transplantation: From microbiome to clinic. Cell Host Microbe31, 712-733 (2023). https://doi.org/10.1016/j.chom.2023.03.020
Hurley, D., McCusker, M. P., Fanning, S. & Martins, M. Salmonella-host interactions - modulation of the host innate immune system. Front Immunol5, 481 (2014). https://doi.org/10.3389/fimmu.2014.00481

No competing interests reported.

westernblot.docx

Download PDF

Editor assigned by journal
16 Jul, 2024
Submission checks completed at journal
16 Jul, 2024
First submitted to journal
02 Jun, 2024

You are reading this latest preprint version

Overexpression of tlr5 affects gut microbiota resistance to Salmonella infection

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Results

Downregulation of Colonic TLR5 Receptor Content in Mice upon Salmonella Infection

Impact of TLR5 Overexpression on Salmonella Infection Outcome

Exploring the Gut Microbiota Diversity through 16S Sequencing Analysis

Non-targeted Metabolomics Reveals Differential Fecal Metabolite Profiles in TLR5 Overexpressing Mice

Methods

Mice

Euthanasia Methods:

Salmonella

Tissue collection and RNA extraction

Real-time quantitative polymerase chain reaction (qPCR) analysis

Western blot analysis

Gut Microbiota Transfer

Intestinal content collection and DNA extraction

16S rRNA gene sequencing analysis

Non-target metabolome sequencing analysis

Data analysis

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1