The development of lactic acid bacteria as mucosal vaccine vectors requires the identification of robust mucosal adjuvants to increase vaccine effectiveness. The E. coli type I fimbriae adhesion protein FimH is of interest as a mucosal adjuvant as it targets microfold (M) cells enhancing vaccine uptake into Peyer’s patches and can activate the innate immune system via Toll-like receptor (TLR) 4 binding. Here, we displayed the N-terminal domain of FimH on the surface of a Lactobacillus acidophilus vaccine vector and evaluated its ability to increase uptake of L. acidophilus into Peyer’s patches and activate innate immune responses. FimH was robustly displayed on the L. acidophilus surface but did not increase uptake into the Peyer’s patches. FimH did increase trafficking of L. acidophilus to mesenteric lymph nodes by antigen-presenting cells including macrophages and dendritic cells. It also increased transcription of retinaldehyde dehydrogenase and decreased transcription of IL-21 in the Peyer’s patches and mesenteric lymph nodes. The N-terminal domain of FimH did not activate TLR4 in vitro, indicating that FimH may stimulate innate immune responses through a not-yet-identified mechanism. These results indicate that E. coli FimH alters the innate immune response to L. acidophilus and should be further studied as an adjuvant for lactic acid bacterial vaccine platforms.

Background: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains the leading cause of death from a single infectious agent worldwide. Exposure to Mtb results in diverse outcomes: bacterial clearance, latent infection, asymptomatic, or symptomatic TB. Current diagnostic tools cannot reliably distinguish these outcomes. Our previous studies identified Mtb proteins in extracellular vesicles (EVs) from TB patients' serum, suggesting their potential as biomarkers. Here, we aimed to discover Mtb proteins and peptides in serum EVs across TB stages, focusing on asymptomatic individuals. Methods: Serum was obtained from healthy, HIV-negative South African adult volunteers enrolled in a TB risk study. Based on patients' outcomes, samples were classified as prevalent, incident (controls that progressed to TB), activated TB, or community controls. EVs were isolated using ExoQuick™, followed by protein digestion and mass spectrometry (MS) analysis. Data-independent acquisition (DIA) with five different data analysis strategies, and two data-dependent (DDA) methods were used to identify Mtb proteins. Results: Our DIA analysis using ion-mobility and spectral libraries enriched with Mtb-MS data revealed 19 Mtb proteins. Rv2997 was significantly higher at baseline in individuals who were initially TB-negative (incident) but later became bacteriologically positive, asymptomatic-TB (activated). HspX, GroEL2, and GroES, together with a MtrB peptide were significantly different between controls and asymptomatic-TB cases. DDA approaches did not resolve additional Mtb proteins. Conclusions: Quantitative DIA analysis discovered Mtb proteins/peptides in serum-derived EVs that were differentially abundant in individuals with early, asymptomatic TB. These findings highlight their potential as biomarkers and provide insight into host–pathogen interactions during subclinical infection.