Protein and Microbial Biomarkers in Sputum Discern Acute and Latent Tuberculosis in Investigation of Pastoral Ethiopian Cohort

Abstract

Differential diagnosis of tuberculosis (TB) and latent TB infection (LTBI) remains a public health priority in high TB burden countries. Pulmonary TB is diagnosed by sputum smear microscopy, chest X-rays, and PCR tests for distinct Mycobacterium tuberculosis (Mtb) genes. Clinical tests to diagnose LTBI rely on immune cell stimulation in blood plasma with TB-specific antigens followed by measurements of interferon-γ concentrations. The latter is an important cytokine for cellular immune responses against Mtb in infected lung tissues. Sputum smear microscopy and chest X-rays are not sufficiently sensitive while both PCR and interferon-γ release assays are expensive. Alternative biomarkers for the development of diagnostic tests to discern TB disease states are desirable. This study’s objective was to discover sputum diagnostic biomarker candidates from the analysis of samples from 161 human subjects including TB patients, individuals with LTBI, negative community controls (NCC) from the province South Omo, a pastoral region in Ethiopia. We analyzed 16S rRNA gene-based bacterial taxonomies and proteomic profiles. The sputum microbiota did not reveal statistically significant differences in α-diversity comparing the cohorts. The genus Mycobacterium, representing Mtb, was only identified for the TB group which also featured reduced abundance of the genus Rothia in comparison with the LTBI and NCC groups. Rothia is a respiratory tract commensal and may be sensitive to the inflammatory milieu generated by infection with Mtb. Proteomic data supported innate immune responses against the pathogen in subjects with pulmonary TB. Ferritin, an iron storage protein released by damaged host cells, was markedly increased in abundance in TB sputum compared to the LTBI and NCC groups, along with the α-1-acid glycoproteins ORM1 and ORM2. These proteins are acute phase reactants and inhibit excessive neutrophil activation. Proteomic data highlight the effector roles of neutrophils in the anti-Mtb response which was not observed for LTBI cases. Less abundant in the sputum of the LTBI group, compared to the NCC group, were two immunomodulatory proteins, mitochondrial TSPO and the extracellular ribonuclease T2. If validated, these proteins are of interest as new biomarkers for diagnosis of LTBI.

Human Sputum Proteomics: Advancing Non-Invasive Diagnosis of Respiratory Diseases with Enhanced Biomarker Analysis Methods

“…It is apparent that the protein composition of sputum best reflects the state of lungs [48]. Additional information on sputum proteomics studies can be found in references [72][73][74][75][76][77][78][79][80]. Aptamer-based assay (SOMAscan) 1129 [71] These references are selected illustrations of efforts to identify, quantify, and characterize proteins and peptides, including antibiotic peptides, present in sputum samples.…”

From simple to complex: Protein‐based biomarker discovery in tuberculosis

“…In a few studies, saliva [14,26,29,30] or sputum [7,[30][31][32][33][34] were explored for human biomarker detection (Table 1). One study of saliva using MS reached the TPP criteria of a triage test [26] (Table 2) using a five-protein biosignature, including alpha-1-antichymotrypsin (SERPINA3), NAD(P)H-hydrate epimerase (NAXE), proteasome subunit beta type-6 (PSMB6), immunoglobulin kappa variable domain 1-33 (IGKV1-33), and neuroserpin (SERPIN1).…”

“…1B). However, a few studies have used it together with other types of omics data [25,31,38,[62][63][64]. Two of the studies [25,63] identified signatures that fulfilled the TPP for a triage test for diagnosing TB disease.…”

“…We further discuss different sample types, experimental platforms, and briefly the signaling pathways associated with the identified host-derived proteins. [12,13], [17], [29], [63] # , [66], [103][104][105][106][107][108][109][110][111][112][113][114] Mass spectrometry [22][23][24], [25] # , [115][116][117][118][119][120][121][122][123][124][125][126][127]] SOMAscan [19,20] Other [21], [70], [78], [128,129] Sputum/Saliva ELISA [7] Multiplex bead assay [14], [29] Mass spectrometry [26], [30], [31] # , [32]<...…”

Crosstalk of pathogens with human immune system in airway mucus profiled via machine learning-enhanced data-independent acquisition mass spectrometry

“…Most often, microbial organisms produce a much lower biomass in sputum in comparison to that of the host. Therefore, detection of most expressed microbial proteins via LC-MS/MS can be challenging, as demonstrated in a study that targeted detection of Mycobacterium tuberculosis from sputum in infected patients by 16S rRNA sequencing as well as LC-MS/MS (HaileMariam et al, 2021). Wu et al reported on the proteomes of Pseudomonas aeruginosa, a common lung pathogen of cystic fibrosis patients using MS-DDA analyses.…”