Selection for antibiotic resistance is reduced when embedded in a natural microbial community, bioRxiv, 2019-01-24
AbstractAntibiotic resistance has emerged as one of the most pressing, global threats to public health. In single-species experiments selection for antibiotic resistance occurs at very low antibiotic concentrations. However, it is unclear how far these findings can be extrapolated to natural environments, where species are embedded within complex communities. We competed isogenic strains of Escherichia coli, differing exclusively in a single chromosomal resistance determinant, in the presence and absence of a pig fecal microbial community across a gradient of antibiotic concentration for two relevant antibiotics gentamicin and kanamycin. We show that the minimal selective concentration was increased by more than one order of magnitude for both antibiotics when embedded in the community. We identified two general mechanisms were responsible for the increase in minimal selective concentration an increase in the cost of resistance and a protective effect of the community for the susceptible phenotype. These findings have implications for our understanding of the evolution and selection of antibiotic resistance, and can inform future risk assessment efforts on antibiotic concentrations.
biorxiv microbiology 100-200-users 2019In vivo structure of the Legionella type II secretion system by electron cryotomography Supplementary material, bioRxiv, 2019-01-19
The type II secretion system (T2SS) is a multi-protein envelope-spanning assembly that translocates a wide range of virulence factors, enzymes and effectors through the outer membrane (OM) of many Gram-negative bacteria. Here, using electron cryotomography and subtomogram averaging methods, we present the first in situ structure of an intact T2SS, imaged within the human pathogen Legionella pneumophila. Although the T2SS has only limited sequence and component homology with the evolutionarily-related Type IV pilus (T4P) system, we show that their overall architectures are remarkably similar. Despite similarities, there are also differences, including for instance that the T2SS-ATPase complex is usually present but disengaged from the inner membrane, the T2SS has a much longer periplasmic vestibule, and it has a short-lived flexible pseudopilus. Placing atomic models of the components into our ECT map produced a complete architectural model of the intact T2SS that provides new insights into the structure and function of its components, its position within the cell envelope, and the interactions between its different subcomplexes. Overall, these structural results strongly support the piston model for substrate extrusion.
biorxiv microbiology 0-100-users 2019Epigenetic suppression of interferon lambda receptor expression leads to enhanced HuNoV replication in vitro, bioRxiv, 2019-01-17
Human norovirus (HuNoV) is the main cause of gastroenteritis worldwide yet no therapeutics are currently available. Here, we utilize a human norovirus replicon in human gastric tumor (HGT) cells to identify host factors involved in promoting or inhibiting HuNoV replication. We observed that an IFN-cured population of replicon-harboring HGT cells (HGT-cured) was enhanced in their ability to replicate transfected HuNoV RNA compared to parental HGT cells, suggesting that differential gene expression in HGT-cured cells created an environment favouring norovirus replication. Microarray analysis was used to identify genes differentially regulated in HGT-NV and HGT-cured compared to parental HGT cells. We found that the IFN lambda receptor alpha (IFNLR1) expression was highly reduced in HGT-NV and HGT-cured cells. All three cell lines responded to exogenous IFN-β by inducing interferon stimulated genes (ISGs), however, HGT-NV and HGT-cured failed to respond to exogenous IFN-λ. Inhibition of DNA methyltransferase activity with 5-aza-2'-deoxycytidine partially reactivated IFNLR1 expression in HGT-NV and IFN-cured cells suggesting that host adaptation occurred via epigenetic reprogramming. In line with this, ectopic expression of the IFN-λ receptor alpha rescued HGT-NV and HGT-cured cells response to IFN-λ. We conclude that type III IFN is important in inhibiting HuNoV replication in vitro and that the loss of IFNLR1 enhances replication of HuNoV. This study unravels for the first time epigenetic reprogramming of the interferon lambda receptor as a new mechanism of cellular adaptation during long-term RNA virus replication and shows that an endogenous level of interferon lambda signalling is able to control human norovirus replication.
biorxiv microbiology 0-100-users 2019Large-scale analyses of human microbiomes reveal thousands of small, novel genes and their predicted functions, bioRxiv, 2018-12-14
AbstractSmall proteins likely abound in prokaryotes, and may mediate much of the communication that occurs between organisms within a microbiome and their host. Unfortunately, small proteins are traditionally overlooked in biology, in part due to the computational and experimental difficulties in detecting them. To systematically identify novel small proteins, we carried out a large comparative genomics study on 1,773 HMP human-associated metagenomes from four different body sites (mouth, gut, skin and vagina). We describe more than four thousand conserved protein families, the majority of which are novel; ~30% of these protein families are predicted to be secreted or transmembrane. Over 90% of the small protein families have no known domain, and almost half are not represented in reference genomes, emphasizing the incompleteness of knowledge in this space. Our analysis exposes putative novel ‘housekeeping’ small protein families, including a potential novel ribosomally associated protein, as well as ‘mammalian-specific’ or ‘human-specific’ protein families. By analyzing the genomic neighborhood of small genes, we pinpoint a subset of families that are potentially associated with defense against bacteriophage. Finally, we identify families that may be subject to horizontal transfer and are thus potentially involved in adaptation of bacteria to the changing human environment. Our study suggest that small proteins are highly abundant and that those of the human microbiome, in particular, may perform diverse functions that have not been previously reported.
biorxiv microbiology 0-100-users 2018Real-time capture of horizontal gene transfers from gut microbiota by engineered CRISPR-Cas acquisition, bioRxiv, 2018-12-11
AbstractHorizontal gene transfer (HGT) is central to the adaptation and evolution of bacteria. However, our knowledge about the flow of genetic material within complex microbiomes is lacking; most studies of HGT rely on bioinformatic analyses of genetic elements maintained on evolutionary timescales or experimental measurements of phenotypically trackable markers (e.g. antibiotic resistance). Consequently, our knowledge of the capacity and dynamics of HGT in complex communities is limited. Here, we utilize the CRISPR-Cas spacer acquisition process to detect HGT events from complex microbiota in real-time and at nucleotide resolution. In this system, a recording strain is exposed to a microbial sample, spacers are acquired from foreign transferred elements and permanently stored in genomic CRISPR arrays. Subsequently, sequencing and analysis of these spacers enables identification of the transferred elements. This approach allowed us to quantify transfer frequencies of individual mobile elements without the need for phenotypic markers or post-transfer replication. We show that HGT in human clinical fecal samples can be extensive and rapid, often involving multiple different plasmid types, with the IncX type being the most actively transferred. Importantly, the vast majority of transferred elements did not carry readily selectable phenotypic markers, highlighting the utility of our approach to reveal previously hidden real-time dynamics of mobile gene pools within complex microbiomes.
biorxiv microbiology 100-200-users 2018Assignment of virus and antimicrobial resistance genes to microbial hosts in a complex microbial community by combined long-read assembly and proximity ligation, bioRxiv, 2018-12-09
AbstractThe characterization of microbial communities by metagenomic approaches has been enhanced by recent improvements in short-read sequencing efficiency and assembly algorithms. We describe the results of adding long-read sequencing to the mix of technologies used to assemble a highly complex cattle rumen microbial community, and compare the assembly to current short read-based methods applied to the same sample. Contigs in the long-read assembly were 7-fold longer on average, and contained 7-fold more complete open reading frames (ORF), than the short read assembly, despite having three-fold lower sequence depth. The linkages between long-read contigs, provided by proximity ligation data, supported identification of 188 novel viral-host associations in the rumen microbial community that suggest cross-species infectivity of specific viral strains. The improved contiguity of the long-read assembly also identified 94 antimicrobial resistance genes, compared to only seven alleles identified in the short-read assembly. Overall, we demonstrate a combination of experimental and computational methods that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.
biorxiv microbiology 0-100-users 2018