Abstract

While changes in gut microbial populations have been described in human immuno-deficiency virus (HIV)-infected patients undergoing antiretroviral therapy (ART), the mechanisms underlying the contributions of gut bacteria and their molecular agents (metabolites and proteins) to immune recovery remain unexplored. To study this, we examined the active fraction of the gut microbiome, through examining protein synthesis and accumulation of metabolites inside gut bacteria and in the bloodstream, in 8 healthy controls and 29 HIV-infected individuals (6 being longitudinally studied). We found that HIV infection is associated to dramatic changes in the active set of gut bacteria simultaneously altering the metabolic outcomes. Effects were accentuated among immunological ART responders, regardless diet, subject characteristics, clinical variables other than immune recovery, the duration and type of ART and sexual preferences. The effect was found at quantitative levels of several molecular agents and active bacteria which were herein identified and whose abundance correlated with HIV immune pathogenesis markers. Although, we cannot rule out the possibility that some changes are partially a random consequence of the disease status, our data suggest that most likely reduced inflammation and immune recovery is a joint solution orchestrated by both the active fraction of the gut microbiota and the host.

Abstract

Background The massive characterization of host-associated and environmental microbial communities has represented a real breakthrough in the life sciences in the last years. In this context, metaproteomics specifically enables the transition from assessing the genomic potential to actually measuring the functional expression of a microbiome. However, significant research efforts are still required to develop analysis pipelines optimized for metaproteome characterization. Results This work presents an efficient analytical pipeline for shotgun metaproteomic analysis, combining bead-beating/freeze-thawing for protein extraction, filter-aided sample preparation for cleanup and digestion, and single-run liquid chromatography-tandem mass spectrometry for peptide separation and identification. The overall procedure is more time-effective and less labor-intensive when compared to state-of-the-art metaproteomic techniques. The pipeline was first evaluated using mock microbial mixtures containing different types of bacteria and yeasts, enabling the identification of up to over 15,000 non-redundant peptide sequences per run with a linear dynamic range from 104 to 108 colony-forming units. The pipeline was then applied to the mouse fecal metaproteome, leading to the overall identification of over 13,000 non-redundant microbial peptides with a false discovery rate of "1%, belonging to over 600 different microbial species and 250 functionally relevant protein families. An extensive mapping of the main microbial metabolic pathways actively functioning in the gut microbiome was also achieved. Conclusions The analytical pipeline presented here may be successfully used for the in-depth and time-effective characterization of complex microbial communities, such as the gut microbiome, and represents a useful tool for the microbiome research community. Electronic supplementary material The online version of this article (doi:10.1186/s40168-014-0049-2) contains supplementary material, which is available to authorized users. Document type: Article