Widespread colonization of the lung by Tropheryma whipplei in HIV infection

Abstract

Rationale: Lung infections caused by opportunistic or virulent pathogens are a principal cause of morbidity and mortality in HIV infection. It is unknown whether HIV infection leads to changes in basal lung microflora, which may contribute to chronic pulmonary complications that increasingly are being recognized in individuals infected with HIV.

Objectives: To determine whether the immunodeficiency associated with HIV infection resulted in alteration of the lung microbiota.

Methods: We used 16S ribosomal RNA targeted pyrosequencing and shotgun metagenomic sequencing to analyze bacterial gene sequences in bronchoalveolar lavage (BAL) and mouths of 82 HIV-positive and 77 HIV-negative subjects.

Measurements and main results: Sequences representing Tropheryma whipplei, the etiologic agent of Whipple's disease, were significantly more frequent in BAL of HIV-positive compared with HIV-negative individuals. T. whipplei dominated the community (>50% of sequence reads) in 11 HIV-positive subjects, but only 1 HIV-negative individual (13.4 versus 1.3%; P = 0.0018). In 30 HIV-positive individuals sampled longitudinally, antiretroviral therapy resulted in a significantly reduced relative abundance of T. whipplei in the lung. Shotgun metagenomic sequencing was performed on eight BAL samples dominated by T. whipplei 16S ribosomal RNA. Whole genome assembly of pooled reads showed that uncultured lung-derived T. whipplei had similar gene content to two isolates obtained from subjects with Whipple's disease.

Conclusions: Asymptomatic subjects with HIV infection have unexpected colonization of the lung by T. whipplei, which is reduced by effective antiretroviral therapy and merits further study for a potential pathogenic role in chronic pulmonary complications of HIV infection.

Figures

Figure 1.

Clustering of data from all study sites. The result of applying principal coordinate analysis to a matrix of unweighted UniFrac distances, showing the relationship between the overall collection of bacterial sequences detected in samples from all of the Lung HIV Microbiome Project (LHMP) cohorts, and that the overall pattern is not driven by methodological differences that existed across cohorts. (A) and (B) are the same plot, but the points are colored by sample type in (A) and by cohort in (B). The control group in (A) is a range of methodological controls, as detailed in the online supplement. PC = principal coordinate.

Figure 2.

Quantitative PCR analysis with the hsp65 gene. (A) Representative PCR of Tropheryma whipplei–specific hsp65 showing a base pair amplicon for a bronchoalveolar lavage (BAL) fluid sample (006–0006) from an individual with high T. whipplei relative abundance based on 16S ribosomal RNA (rRNA) in the Colorado–San Francisco (SF) cohort. (B) Copy numbers normalized to 16S rRNA expression in the Colorado, Indiana, and Pennsylvania cohorts is shown.

Figure 3.

Longitudinal analysis of Tropheryma whipplei relative abundance in individuals on antiretroviral therapy (ART) treatment. The relative abundance of T. whipplei is plotted for the six individuals in whom the T. whipplei phylotype exceeded 10% of the sequences in at least one sample. Columns denoted with “ND” had no data, either because the 3-year time point was not available or because the sample did not achieve a sequencing depth of at least 225 reads.

Figure 4.

Gene content of lung Tropheryma whipplei strains deduced from metagenomic data. The outer ring shows the TW08/27 genome, the next inner circle shows the genes missing in Twist, followed by the genes missing in lung pooled assembly. The inner circle shows the 14 WiSP loci in the TW0827 reference. The gene content plot shows above-average regions in olive and the below-average regions in purple. The color code for the outer ring indicates functional categories for gene: dark blue, pathogenicity or adaptation; black, energy metabolism; red, information transfer; dark green, membranes or surface structures; cyan, degradation of macromolecules; purple, degradation of small molecules; yellow, central or intermediary metabolism; light blue, regulators; orange, conserved hypothetical; pale green, unknown; and brown, pseudogenes.