Nutritional markers and proteome in patients undergoing treatment for pulmonary tuberculosis differ by geographic region

Abstract

Introduction: Contemporary phase 2 TB disease treatment clinical trials have found that microbiologic treatment responses differ between African versus non-African regions, the reasons for which remain unclear. Understanding host and disease phenotypes that may vary by region is important for optimizing curative treatments.

Methods: We characterized clinical features and the serum proteome of phase 2 TB clinical trial participants undergoing treatment for smear positive, culture-confirmed TB, comparing host serum protein expression in clinical trial participants enrolled in African and Non-African regions. Serum samples were collected from 289 participants enrolled in the Centers for Disease Control and Prevention TBTC Study 29 (NCT00694629) at time of enrollment and at the end of the intensive phase (after 40 doses of TB treatment).

Results: After a peptide level proteome analysis utilizing a unique liquid chromatography IM-MS platform (LC-IM-MS) and subsequent statistical analysis, a total of 183 core proteins demonstrated significant differences at both baseline and at week 8 timepoints between participants enrolled from African and non-African regions. The majority of the differentially expressed proteins were upregulated in participants from the African region, and included acute phase proteins, mediators of inflammation, as well as coagulation and complement pathways. Downregulated proteins in the African population were primarily linked to nutritional status and lipid metabolism pathways.

Conclusions: We have identified differentially expressed nutrition and lipid pathway proteins by geographic region in TB patients undergoing treatment for pulmonary tuberculosis, which appear to be associated with differential treatment responses. Future TB clinical trials should collect expanded measures of nutritional status and further evaluate the relationship between nutrition and microbiologic treatment response.

Conflict of interest statement

The authors declare no competing interests in regard any relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, including those authors currently affiliated with Merck & Co Inc. and Meso Scale Diagnostics.

Figures

Fig 1. Comparison of African and non-African serum plasma proteome data.

A) Schematic view of study sites and participants. Serum proteome data was compared at B) baseline and C) after eight weeks of treatment across 270 patients to determine the significant proteins, p value <0.05, that differentiate African versus Non-African patients. Disease severity is representing by stratification by cavitary size. 219 and 240 proteins were identified respectively from these time points which resulted in an overlap of D) 183 proteins, which forms a core protein signature of African/Non-African differentiation independent of treatment.

Fig 2. Characterization of the 183 African/non-African discriminatory core protein signature.

Shown in A) is the fold change difference in abundance between African and non-African TB patient population for each of the 183 proteins in rank order. Negative, lower, fold change is in reference to the African population. Asterisks mark two proteins SHBG and TIMP1, which change directionality between baseline and week 8 results, where all other proteins show similar directionally of change between these two time point datasets. B) Gene Oncology (GO) annotations with Bonferroni adjusted p-values generated via DAVID show those pathways downregulated in African populations. C) Protein pathways upregulated in African populations.

Fig 3. Boxplot of representative proteins across cavitary size.

A-D) Boxplot representation of quantitative global MS data from baseline values for two proteins representative of lipid and nutrient transport downregulation within the African cohort and two proteins representing acute inflammatory activation upregulated with the African cohort, all stratified across cavitary size representing initial disease severity.

Fig 4. Comparison of apolipoprotein plasma signature with BMI and culture status.

A) Heatmap, Pearson correlation, of all apolipoproteins encompassed within the African TB patient discriminatory protein signature stratified across cohort, timepoint, and BMI. Overweight >25 BMI, Healthy weight between 18.5 and 24.9 BMI, Underweight, <18.5 BMI. B) Graph of the apolipoprotein signature from the 8 proteins downregulated in the African cohort, stratified across BMI range. C) Graph of the apolipoprotein signature stratified across 8 week culture conversion status. B) and C) Values represent median protein abundances from a scaled normalization within each protein across time point and cohort. Error bars represent the standard deviation across the median protein values.

Fig 5. Comparison of inflammatory and complement activation plasma signature.

A) Heatmap, Pearson correlation, of proteins found in the acute inflammatory response and complement activation pathways from Fig 2C, with redundancy removed. Results are stratified across cohort, timepoint, and disease severity as determined by cavitary size by baseline chest radiograph. B) Graph of the composite (14 proteins from panel A) acute inflammatory protein signature across timepoint and disease severity C) Graph of the composite (19 proteins from panel A) complement activation protein signature across timepoint and disease severity. Values in panel B) and C) represent median protein abundances from a scaled normalization within each protein across time point and cohort. Error bars represent the standard deviation across the median protein values.

Fig 6. Comparing retinol binding protein and transthyretin abundances to understand inflammation and vitamin A deficiency in African TB patients.

A) graph combining Retinal Binding Protein (RET4/RBP) and Transthyretin (TTHY) abundance changes between cohorts. B) Graph of RET4 only, stratified across both cohorts and patient culture status (positive encompasses both liquid and solid) at 8 weeks as a measure of outcome. C) Graph of TTHY only, stratified across both cohorts and patient culture status. Error bars for all panels represents the variance across patient population for each protein and time point.