Bidirectional transfer of Anelloviridae lineages between graft and host during lung transplantation.

Solid organ transplantation disrupts virus-host relationships, potentially resulting in viral transfer from donor to recipient, reactivation of latent viruses, and new viral infections. Viral transfer, colonization, and reactivation are typically monitored using assays for specific viruses, leaving the behavior of full viral populations (the "virome") understudied. Here we sought to investigate the temporal behavior of viruses from donor lungs and transplant recipients comprehensively. We interrogated the bronchoalveolar lavage and blood viromes during the peritransplant period and 6-16 months posttransplant in 13 donor-recipient pairs using shotgun metagenomic sequencing. Anelloviridae, ubiquitous human commensal viruses, were the most abundant human viruses identified. Herpesviruses, parvoviruses, polyomaviruses, and bacteriophages were also detected. Anelloviridae populations were complex, with some donor organs and hosts harboring multiple contemporaneous lineages. We identified transfer of Anelloviridae lineages from donor organ to recipient serum in 4 of 7 cases that could be queried, and immigration of lineages from recipient serum into the allograft in 6 of 10 such cases. Thus, metagenomic analyses revealed that viral populations move between graft and host in both directions, showing that organ transplantation involves implantation of both the allograft and commensal viral communities.

Transcriptomic and proteomic insights into innate immunity and adaptations to a symbiotic lifestyle in the gutless marine worm Olavius algarvensis.

BACKGROUND: The gutless marine worm Olavius algarvensis has a completely reduced digestive and excretory system, and lives in an obligate nutritional symbiosis with bacterial symbionts. While considerable knowledge has been gained of the symbionts, the host has remained largely unstudied. Here, we generated transcriptomes and proteomes of O. algarvensis to better understand how this annelid worm gains nutrition from its symbionts, how it adapted physiologically to a symbiotic lifestyle, and how its innate immune system recognizes and responds to its symbiotic microbiota. RESULTS: Key adaptations to the symbiosis include (i) the expression of gut-specific digestive enzymes despite the absence of a gut, most likely for the digestion of symbionts in the host's epidermal cells; (ii) a modified hemoglobin that may bind hydrogen sulfide produced by two of the worm's symbionts; and (iii) the expression of a very abundant protein for oxygen storage, hemerythrin, that could provide oxygen to the symbionts and the host under anoxic conditions. Additionally, we identified a large repertoire of proteins involved in interactions between the worm's innate immune system and its symbiotic microbiota, such as peptidoglycan recognition proteins, lectins, fibrinogen-related proteins, Toll and scavenger receptors, and antimicrobial proteins. CONCLUSIONS: We show how this worm, over the course of evolutionary time, has modified widely-used proteins and changed their expression patterns in adaptation to its symbiotic lifestyle and describe expressed components of the innate immune system in a marine oligochaete. Our results provide further support for the recent realization that animals have evolved within the context of their associations with microbes and that their adaptive responses to symbiotic microbiota have led to biological innovations.

Strain-resolved microbial community proteomics reveals simultaneous aerobic and anaerobic function during gastrointestinal tract colonization of a preterm infant.

While there has been growing interest in the gut microbiome in recent years, it remains unclear whether closely related species and strains have similar or distinct functional roles and if organisms capable of both aerobic and anaerobic growth do so simultaneously. To investigate these questions, we implemented a high-throughput mass spectrometry-based proteomics approach to identify proteins in fecal samples collected on days of life 13-21 from an infant born at 28 weeks gestation. No prior studies have coupled strain-resolved community metagenomics to proteomics for such a purpose. Sequences were manually curated to resolve the genomes of two strains of Citrobacter that were present during the later stage of colonization. Proteome extracts from fecal samples were processed via a nano-2D-LC-MS/MS and peptides were identified based on information predicted from the genome sequences for the dominant organisms, Serratia and the two Citrobacter strains. These organisms are facultative anaerobes, and proteomic information indicates the utilization of both aerobic and anaerobic metabolisms throughout the time series. This may indicate growth in distinct niches within the gastrointestinal tract. We uncovered differences in the physiology of coexisting Citrobacter strains, including differences in motility and chemotaxis functions. Additionally, for both Citrobacter strains we resolved a community-essential role in vitamin metabolism and a predominant role in propionate production. Finally, in this case study we detected differences between genome abundance and activity levels for the dominant populations. This underlines the value in layering proteomic information over genetic potential.

Metaproteomics reveals functional shifts in microbial and human proteins during a preterm infant gut colonization case.

Microbial colonization of the human gastrointestinal tract plays an important role in establishing health and homeostasis. However, the time-dependent functional signatures of microbial and human proteins during early colonization of the gut have yet to be determined. To this end, we employed shotgun proteomics to simultaneously monitor microbial and human proteins in fecal samples from a preterm infant during the first month of life. Microbial community complexity increased over time, with compositional changes that were consistent with previous metagenomic and rRNA gene data. More specifically, the function of the microbial community initially involved biomass growth, protein production, and lipid metabolism, and then switched to more complex metabolic functions, such as carbohydrate metabolism, once the community stabilized and matured. Human proteins detected included those responsible for epithelial barrier function and antimicrobial activity. Some neutrophil-derived proteins increased in abundance early in the study period, suggesting activation of the innate immune system. Likewise, abundances of cytoskeletal and mucin proteins increased later in the time course, suggestive of subsequent adjustment to the increased microbial load. This study provides the first snapshot of coordinated human and microbial protein expression in a preterm infant's gut during early development.

Metaproteomics reveals abundant transposase expression in mutualistic endosymbionts.

Transposases, enzymes that catalyze the movement of mobile genetic elements, are the most abundant genes in nature. While many bacteria encode an abundance of transposases in their genomes, the current paradigm is that the expression of transposase genes is tightly regulated and generally low due to its severe mutagenic effects. In the current study, we detected the highest number of transposase proteins ever reported in bacteria, in symbionts of the gutless marine worm Olavius algarvensis with metaproteomics. At least 26 different transposases from 12 different families were detected, and genomic and proteomic analyses suggest that many of these are active. This high expression of transposases indicates that the mechanisms for their tight regulation have been disabled or no longer exist.

Phage-induced expression of CRISPR-associated proteins is revealed by shotgun proteomics in Streptococcus thermophilus.

The CRISPR/Cas system, comprised of clustered regularly interspaced short palindromic repeats along with their associated (Cas) proteins, protects bacteria and archaea from viral predation and invading nucleic acids. While the mechanism of action for this acquired immunity is currently under investigation, the response of Cas protein expression to phage infection has yet to be elucidated. In this study, we employed shotgun proteomics to measure the global proteome expression in a model system for studying the CRISPR/Cas response in S. thermophilus DGCC7710 infected with phage 2972. Host and viral proteins were simultaneously measured following inoculation at two different multiplicities of infection and across various time points using two-dimensional liquid chromatography tandem mass spectrometry. Thirty-seven out of forty predicted viral proteins were detected, including all proteins of the structural virome and viral effector proteins. In total, 1,013 of 2,079 predicted S. thermophilus proteins were detected, facilitating the monitoring of host protein synthesis changes in response to virus infection. Importantly, Cas proteins from all four CRISPR loci in the S. thermophilus DGCC7710 genome were detected, including loci previously thought to be inactive. Many Cas proteins were found to be constitutively expressed, but several demonstrated increased abundance following infection, including the signature Cas9 proteins from the CRISPR1 and CRISPR3 loci, which are key players in the interference phase of the CRISPR/Cas response. Altogether, these results provide novel insights into the proteomic response of S. thermophilus, specifically CRISPR-associated proteins, upon phage 2972 infection.

neonatally primed lymph node, but not splenic T cells, display a Gly-Gly motif within the TCR beta-chain complementarity-determining region 3 that controls affinity and may affect lymphoid organ retention.

Ig-proteolipid protein 1 (Ig-PLP1) is an Ig chimera expressing the encephalitogenic PLP1 peptide corresponding to amino acid residues 139-151 of PLP. Newborn mice given Ig-PLP1 in saline on the day of birth and challenged 7 wk later with PLP1 peptide in CFA develop an organ-specific neonatal immunity that confers resistance against experimental allergic encephalomyelitis. The T cell responses in these animals are comprised of Th2 cells in the lymph node and anergic Th1 lymphocytes in the spleen. Intriguingly, the anergic splenic T cells, although nonproliferative and unable to produce IFN-gamma or IL-4, secrete significant amounts of IL-2. Studies were performed to determine whether the two populations display any structural differences in the TCR H chain variable region that could contribute to the differential affinity and retention in different organs. Responsive Th2 lymph node T cells and anergic splenic lymphocytes were immortalized, and the structures of their TCR Vbeta were determined. The results show that Vbeta and Jbeta usage was random, but the CDR3 regions of the lymph node cells had a conserved Gly-Gly motif. Analysis of TCR affinity/avidity correlated the Gly-Gly motif with lower affinity and retention of the Th2 cells in the lymph node. Also, it is suggested that a higher TCR affinity may be a contributing factor for the development of the neonatal Th1 response in the spleen.