Correlation of the lung microbiota with metabolic profiles in bronchoalveolar lavage fluid in HIV infection

Abstract

Background: While 16S ribosomal RNA (rRNA) sequencing has been used to characterize the lung's bacterial microbiota in human immunodeficiency virus (HIV)-infected individuals, taxonomic studies provide limited information on bacterial function and impact on the host. Metabolic profiles can provide functional information on host-microbe interactions in the lungs. We investigated the relationship between the respiratory microbiota and metabolic profiles in the bronchoalveolar lavage fluid of HIV-infected and HIV-uninfected outpatients.

Results: Targeted sequencing of the 16S rRNA gene was used to analyze the bacterial community structure and liquid chromatography-high-resolution mass spectrometry was used to detect features in bronchoalveolar lavage fluid. Global integration of all metabolic features with microbial species was done using sparse partial least squares regression. Thirty-nine HIV-infected subjects and 20 HIV-uninfected controls without acute respiratory symptoms were enrolled. Twelve mass-to-charge ratio (m/z) features from C18 analysis were significantly different between HIV-infected individuals and controls (false discovery rate (FDR) = 0.2); another 79 features were identified by network analysis. Further metabolite analysis demonstrated that four features were significantly overrepresented in the bronchoalveolar lavage (BAL) fluid of HIV-infected individuals compared to HIV-uninfected, including cystine, two complex carbohydrates, and 3,5-dibromo-L-tyrosine. There were 231 m/z features significantly associated with peripheral blood CD4 cell counts identified using sparse partial least squares regression (sPLS) at a variable importance on projection (VIP) threshold of 2. Twenty-five percent of these 91 m/z features were associated with various microbial species. Bacteria from families Caulobacteraceae, Staphylococcaceae, Nocardioidaceae, and genus Streptococcus were associated with the greatest number of features. Glycerophospholipid and lineolate pathways correlated with these bacteria.

Conclusions: In bronchoalveolar lavage fluid, specific metabolic profiles correlated with bacterial organisms known to play a role in the pathogenesis of pneumonia in HIV-infected individuals. These findings suggest that microbial communities and their interactions with the host may have functional metabolic impact in the lung.

Fig. 1

Principal coordinate analysis (PCoA) plot showing the clustering trend of bronchoalveolar (BAL) samples based on the weighted UniFrac distance. PCoA plot showing the clustering trend of bronchoalveolar (BAL) samples based on the weighted UniFrac distance between all OTUs identified in HIV-infected (n = 39) and HIV-uninfected (n = 20) individuals. In this cohort, the lung microbiota does not distinguish HIV status, but comparison of intra-group beta diversity showed a significant difference in UniFrac distances between the HIV-infected and HIV-uninfected subjects, suggesting a higher degree of heterogeneity among the microbial composition of the lower airways in HIV-infected subjects

Fig. 2

Principal coordinate analysis (PCoA) analysis of 12 m/z features from C18 chromatography. PCoA analysis of 12 m/z features from C18 chromatography shows that the lung metabolome does distinguish HIV-status and there is decreased variability in the metabolome in HIV-infected subjects compared to HIV-uninfected subjects

Fig. 3

Association of peripheral CD4 cell counts with metabolomic features using sPLS at a variable importance on projection (VIP) threshold of 2. Association of peripheral CD4 cell counts with metabolomic features shows that 231 m/z features were found to be significantly associated with CD4 count using partial least squares (PLS) regression at variable importance on projection threshold of 2. Metabolite annotation and pathway enrichment analysis using Mummichog mapped these 231 m/z features to inflammatory pathways, including fatty acid activation and arginine metabolic pathways

Fig. 4

a Three-way relationship between metabolome, HIV, and microbiota integration of HIV-specific m/z features with microbiota data at correlation of 0.3. This figure shows that of the 91 m/z features associated with HIV, 23 were also found to be associated with 29 microbial genera at a correlation of 0.30; red circles microbial families and genus, blue rectangles metabolome features. b Pathway analysis using m/z features that were associated with Staphylococcaceae in the global network. Using Mummichog, metabolite annotation and pathway enrichment analysis was done using the m/z features that were associated with Staphylococcaceae (as the representative bacteria since pathways for Caulobacteraceae and Nocardioidaceae were very similar). The y-axis represents the enrichment significance of the metabolic pathways. This analysis shows that the pathways most affected included lineolate, glycerophospholipid, and fatty acid metabolism

Fig. 5

Overall schema. Overall schematic that postulates that HIV infection alters metabolomic profiles in lung (inflammatory and oxidant pathways) which causes a shift in the functional properties of the lung microbiome and/or in the host response to the lung microbiome which may lead to an increased risk of lung infection or to chronic lung disease associated with HIV