Host-microbiota interactions in mammals

The mammalian gut microbiota provides important functions for the host but at the same time varies greatly between species, populations, individuals and even within individuals over time. Drivers of this variation and particularly their relative importance remains poorly understood. Further, a large proportion of gut microbiota research has been conducted using the laboratory mouse as a model organism. However, laboratory mice inhabit an artificial world where individuals are inbred, the environment is stable, and microbial exposures are limited. As a result of domestication, the gut microbiota of laboratory mice differs in notable ways from that of their wild relatives, and studies investigating the gut microbiota of wild house mice have great potential to aid the interpretation of findings from the laboratory. In this thesis, I explore how the gut microbiota varies between laboratory and wild mice, and examine drivers of natural gut microbiota variation among and within wild mouse populations. I show that although wild mice have a compositionally and functionally more diverse gut microbiota with a faster turnover rate that is clearly shaped by their more complex environment, major patterns of gut microbiota assembly during early life are conserved between laboratory and wild house mice. Since the gut microbiota showed strong age-related variation, I developed a non-invasive epigenetic clock-based method for assessing age in wild mice, whose application showed promise for future studies estimating biological (if not chronological) age in this system. Gut microbiota composition was also influenced by spatial factors, with stronger effects on the fungal than bacterial components of the microbiota, and microbial transmission from soil was identified as one possible source of spatial variation. Finally, I investigate gut microbial ecology in a mammalian species that is quite the opposite of a model organism, the critically endangered Saiga antelope which suffers periodic mass mortality events. I characterise healthy gut microbiota in comparison to other similar species, providing important background for future conservation efforts, as well as contributing to our broader understanding of what shapes microbiota among mammals. Together the results from this thesis increase our understanding of the gut microbiota variation but also highlight challenges in studying the gut microbiota, in the laboratory and beyond

A ProteomicS study on Chlamydia pneumoniae -Induced Changes in Macrophages

Peer reviewe

Assaying Chlamydia pneumoniae Persistence in Monocyte-Derived Macrophages Identifies Dibenzocyclooctadiene Lignans as Phenotypic Switchers

Antibiotic-tolerant persister bacteria involve frequent treatment failures, relapsing infections and the need for extended antibiotic treatment. The virulence of an intracellular human pathogen C. pneumoniae is tightly linked to its propensity for persistence and means for its chemosensitization are urgently needed. In the current work, persistence of C. pneumoniae clinical isolate CV6 was studied in THP-1 macrophages using quantitative PCR and quantitative culture. A dibenzocyclooctadiene lignan schisandrin reverted C. pneumoniae persistence and promoted productive infection. The concomitant administration of schisandrin and azithromycin resulted in significantly improved bacterial eradication compared to sole azithromycin treatment. In addition, the closely related lignan schisandrin C was superior to azithromycin in eradicating the C. pneumoniae infection from the macrophages. The observed chemosensitization of C. pneumoniae was associated with the suppression of cellular glutathione pools by the lignans, implying to a previously unknown aspect of chlamydia–host interactions. These data indicate that schisandrin lignans induce a phenotypic switch in C. pneumoniae, promoting the productive and antibiotic-susceptible phenotype instead of persistence. By this means, these medicinal plant -derived compounds show potential as adjuvant therapies for intracellular bacteria resuscitation

Cohort profile: Finnish Health and Early Life Microbiota (HELMi) longitudinal birth cohort

Peer reviewe

Chlamydia pneumoniae Interferes with Macrophage Differentiation and Cell Cycle Regulation to Promote Its Replication

Chlamydia pneumoniae is a ubiquitous intracellular bacterium which infects humans via the respiratory route. The tendency of C. pneumoniae to persist in monocytes and macrophages is well known, but the underlying host-chlamydial interactions remain elusive. In this work, we have described changes in macrophage intracellular signaling pathways induced by C. pneumoniae infection. Label-free quantitative proteome analysis and pathway analysis tools were used to identify changes in human THP-1-derived macrophages upon C. pneumoniae CV6 infection. At 48-h postinfection, pathways associated to nuclear factor kappa B (NF-kappa B) regulation were stressed, while negative regulation on cell cycle control was prominent at both 48 h and 72 h. Upregulation of S100A8 and S100A9 calcium binding proteins, osteopontin, and purine nucleoside hydrolase, laccase domain containing protein 1 (LACC1) underlined the proinflammatory consequences of the infection, while elevated NF-kappa B2 levels in infected macrophages indicates interaction with the noncanonical NF-kappa B pathway. Infection-induced alteration of cell cycle control was obvious by the downregulation of mini chromosome maintenance (MCM) proteins MCM2-7, and the significance of host cell cycle regulation for C. pneumoniae replication was demonstrated by the ability of a cyclin-dependent kinase (CDK) 4/6 inhibitor Palbociclib to promote C. pneumoniae replication and infectious progeny production. The infection was found to suppress retinoblastoma expression in the macrophages in both protein and mRNA levels, and this change was reverted by treatment with a histone deacetylase inhibitor. The epigenetic suppression of retinoblastoma, along with upregulation of S100A8 and S100A9, indicate host cell changes associated with myeloid-derived suppressor cell (MDSC) phenotype.Peer reviewe

A 16S rRNA gene and draft genome database for the murine oral bacterial community

A curated murine oral microbiome database to be used as a reference for mouse-based studies has been constructed using a combination of bacterial culture, 16S rRNA gene amplicon, and whole-genome sequencing. The database comprises a collection of nearly full-length 16S rRNA gene sequences from cultured isolates and draft genomes from representative taxa collected from a range of sources, including specific-pathogen-free laboratory mice, wild Mus musculus domesticus mice, and formerly wild wood mouse Apodemus sylvaticus. At present, it comprises 103 mouse oral taxa (MOT) spanning four phyla—Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes—including 12 novel undescribed species-level taxa. The key observations from this study are (i) the low diversity and predominantly culturable nature of the laboratory mouse oral microbiome and (ii) the identification of three major murine-specific oral bacterial lineages, namely, Streptococcus danieliae (MOT10), Lactobacillus murinus (MOT93), and Gemella species 2 (MOT43), which is one of the novel, still-unnamed taxa. Of these, S. danieliae is of particular interest, since it is a major component of the oral microbiome from all strains of healthy and periodontally diseased laboratory mice, as well as being present in wild mice. It is expected that this well-characterized database should be a useful resource for in vitro experimentation and mouse model studies in the field of oral microbiology

The influence of dibenzocyclooctadiene lignans on macrophage glutathione and lipid metabolism associated with Chlamydia pneumoniae-induced foam cell formation

Triggered by changes in macrophage redox status and lipid metabolism, foam cells represent a hallmark of atherosclerosis. Induction of macrophage foam cell formation by Chlamydia pneumoniae, a gram-negative human pathogen, has been established in various earlier studies in vitro and in vivo. Oxidation of low-density lipoprotein (LDL) by C. pneumoniae and alterations in macrophage lipid metabolism do not require chlamydial replication, making conventional antibiotics useless in the intervention of the process. In this work, we report on the ability of schisandrin B and schisandrin C, two dibenzocyclooctadiene lignans, to suppress the C. pneumoniae -induced foam cell formation in RAW264.7 macrophages. This effect was accompanied with the upregulation of PPARγ, a nuclear receptor acting as a major transcriptional regulator of lipid metabolism and inflammatory responses. Schisandrin B and schisandrin C also increased the total intracellular glutathione content of the macrophages. In the case of schisandrin B, this was accompanied with the upregulation of GSH biosynthetic genes glutamate cysteine ligase (both the catalytic and the modifier subunits GCLc and GCLm) as well as gamma-glutamyl transpeptidase GGT1. In addition, schisandrin B and schisandrin C upregulated the expression of a lipid transport protein ABCA1 gene mediating cholesterol efflux from macrophages translating into a reduction in total cholesterol concentration in the schisandrin B -treated cells. Collectively, these data indicate that both schisandrin B and schisandrin C are able to alleviate the pathogenic consequences of C. pneumoniae infection in macrophages by altering the cellular redox balance and lipid trafficking.Peer reviewe

Impact of azithromycin, doxycycline and redox-active small molecules on amoxicillin-induced Chlamydia pneumoniae persistence

Amoxicillin is recommended as primary treatment for community-acquired bacterial pneumonia (CABP). 5–10% of CABP cases are caused by Chlamydia pneumoniae, an obligate intracellular bacterium which responds to beta-lactam antibiotics by converting to a persistent phenotype. To support rational pharmacotherapy of C. pneumoniae infections, we investigated how clinically relevant concentrations of azithromycin and doxycycline affect amoxicillin induced C. pneumoniae persistence. Given the known role of redox state alterations in the action of bactericidal antibiotics and widespread use of redox-active dietary supplements when experiencing respiratory symptoms, we also studied how redox active compounds affect the studied antibiotic treatments. Our data demonstrate that clinically applied amoxicillin concentrations (10 and 25 mg/l) fail to eradicate C. pneumoniae infection in respiratory epithelial cells. Transmission electron microscopy (TEM) of amoxicillin-treated C. pneumoniae infected cells reveal aberrant bacterial morphology characteristic of chlamydial stress response. Amoxicillin was also found to significantly limit the antichlamydial effect of azithromycin or doxycycline. However, based on quantitative culture and quantitative PCR data, azithromycin was superior to doxycycline in C. pneumoniae eradication either as monotherapy or in combination with amoxicillin. Amoxicillin was also found to decrease respiratory epithelial cell glutathione (GSH) levels, whereas redox-active dibenzocyclooctadiene lignans increased C. pneumoniae load in amoxicillin-treated cultures up to two-fold. These data highlight the impact of relative administration time on the efficacy of antichlamydial antibiotics and indicate unfavorable interactions between amoxicillin and redox-active small molecules.Peer reviewe

Impact of azithromycin, doxycycline and redox-active small molecules on amoxicillin-induced Chlamydia pneumoniae persistence

Amoxicillin is recommended as primary treatment for community-acquired bacterial pneumonia (CABP). 5–10% of CABP cases are caused by Chlamydia pneumoniae, an obligate intracellular bacterium which responds to beta-lactam antibiotics by converting to a persistent phenotype. To support rational pharmacotherapy of C. pneumoniae infections, we investigated how clinically relevant concentrations of azithromycin and doxycycline affect amoxicillin induced C. pneumoniae persistence. Given the known role of redox state alterations in the action of bactericidal antibiotics and widespread use of redox-active dietary supplements when experiencing respiratory symptoms, we also studied how redox active compounds affect the studied antibiotic treatments. Our data demonstrate that clinically applied amoxicillin concentrations (10 and 25 mg/l) fail to eradicate C. pneumoniae infection in respiratory epithelial cells. Transmission electron microscopy (TEM) of amoxicillin-treated C. pneumoniae infected cells reveal aberrant bacterial morphology characteristic of chlamydial stress response. Amoxicillin was also found to significantly limit the antichlamydial effect of azithromycin or doxycycline. However, based on quantitative culture and quantitative PCR data, azithromycin was superior to doxycycline in C. pneumoniae eradication either as monotherapy or in combination with amoxicillin. Amoxicillin was also found to decrease respiratory epithelial cell glutathione (GSH) levels, whereas redox-active dibenzocyclooctadiene lignans increased C. pneumoniae load in amoxicillin-treated cultures up to two-fold. These data highlight the impact of relative administration time on the efficacy of antichlamydial antibiotics and indicate unfavorable interactions between amoxicillin and redox-active small molecules