Host- and Age-Dependent Transcriptional Changes in Mycobacterium tuberculosis Cell Envelope Biosynthesis Genes after Exposure to Human Alveolar Lining Fluid

Abstract

Tuberculosis (TB) infection, caused by the airborne pathogen Mycobacterium tuberculosis (M.tb), resulted in almost 1.4 million deaths in 2019, and the number of deaths is predicted to increase by 20% over the next 5 years due to the COVID-19 pandemic. Upon reaching the alveolar space, M.tb comes into close contact with the lung mucosa before and after its encounter with host alveolar compartment cells. Our previous studies show that homeostatic, innate soluble components of the alveolar lining fluid (ALF) can quickly alter the cell envelope surface of M.tb upon contact, defining subsequent M.tb–host cell interactions and infection outcomes in vitro and in vivo. We also demonstrated that ALF from 60+ year old elders (E-ALF) vs. healthy 18- to 45-year-old adults (A-ALF) is dysfunctional, with loss of homeostatic capacity and impaired innate soluble responses linked to high local oxidative stress. In this study, a targeted transcriptional assay shows that M.tb exposure to human ALF alters the expression of its cell envelope genes. Specifically, our results indicate that A-ALF-exposed M.tb upregulates cell envelope genes associated with lipid, carbohydrate, and amino acid metabolism, as well as genes associated with redox homeostasis and transcriptional regulators. Conversely, M.tb exposure to E-ALF shows a lesser transcriptional response, with most of the M.tb genes unchanged or downregulated. Overall, this study indicates that M.tb responds and adapts to the lung alveolar environment upon contact, and that the host ALF status, determined by factors such as age, might play an important role in determining infection outcome.

Exposure ofMycobacterium tuberculosisto human alveolar lining fluid shows temporal and strain-specific adaptation to the lung environment

“…In response to this reduction, M.tb exposed to ALF upregulates genes involved in the synthesis of TDM and mannose-containing biomolecules (e.g. ManLAM) (Allue-Guardia et al 2022). However, although these cell envelope biosynthesis genes are quickly upregulated by ALF exposure and their restoration might be associated with the increased uptake and intracellular growth rate trend observed for most of the strains tested, their lasting effects of replenishing TDM and ManLAM on the M.tb surface are still uncertain.…”

“…Collection of BALF and ALF isolation -BALF was collected from healthy adult donors in sterile endotoxin-free saline (0.9% NaCl), filtered through 0.2 μm filters, and concentrated 20-fold using Amicon Ultra Centrifugal Filter Units with a 10-kDa molecular mass cutoff membrane (Millipore Sigma, Burlington, MA, USA) to obtain the ALF physiological concentration within the lung (1 mg/mL of phospholipid), as we described (Arcos et al 2011;Moliva et al 2014;Arcos et al 2015;Arcos et al 2017;Scordo et al 2017;Moliva et al 2019;Scordo et al 2019;Olmo-Fontánez et al 2021;Allue-Guardia et al 2022). ALF was aliquoted and stored at −80 °C until further use.…”

“…M.tb strains CDC1551, H 37 R v (ATCC# 25618), HN878 (BEI Resources, NR-13647), and W-7642 were cultured from frozen stocks in 7H11 agar plates (BD BBL), supplemented with oleic acid, albumin, dextrose and catalase (OADC) for 14 days at 37°C. Single bacterial suspensions (∼ 1 x 10 9 bacteria/ml) were obtained as described (Arcos et al 2011; Arcos et al 2015; Arcos et al 2017; Scordo et al 2017; Moliva et al 2019; Scordo et al 2019; Olmo-Fontánez et al 2021; Allue-Guardia et al 2022), and further exposed to a pool of ALFs (n=17 different donors, 50:50 male:female ratio, no discrimination in ethnicity and race) for 15 min or ∼12 h at 37°C. Using a pool instead of individual ALFs allows us to reduce human variability and focus on the overall effect of ALF in each M.tb strain rather than changes due to ALF-specific factors.…”

“…Briefly, 500 ng of RNA were used for the synthesis of cDNA using the RevertAid H Minus First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) with random hexamer primers, following the manufacturer’s instructions. Then, cDNA and specific primers were used in a 20 μl qPCR reaction with PowerUp SYBR Green Master Mix (Applied Biosystems, Waltham, MA, USA) run in an Applied Biosystems 7500 Real-Time PCR instrument with the following settings: reporter SYBR Green, no quencher, passive reference dye ROX, standard ramp speed, and continuous melt curve ramp increment, as described (Allue-Guardia et al 2022). Relative expression was calculated using the 2 −ΔΔCT method (Livak and Schmittgen 2001) with sigA as the housekeeping gene.…”

Tuberculosis in an Aging World

“…Such studies confirmed that inflammation is not limited to the periphery, but can also be found within the lung and, likely, within other tissues too. Related to M.tb infection control, the ALF of elderly humans can differentially alter the M.tb cell envelope surface upon contact [ 35 , 36 , 37 ], and alter the expression of genes involved in cell envelope remodeling, metabolism, and virulence [ 38 ]. Significantly, M.tb exposure to ALF from the elderly can increase bacterial virulence and pathogenicity in vivo in mice [ 39 ].…”