One-third of the world's population is infected with Mycobacterium tuberculosis (M.tb), which causes tuberculosis. Mycobacterium tuberculosis cell envelope components such as glycolipids, lipoglycans and polysaccharides play important roles in bacteria-host cell interactions that dictate the host immune response. However, little is known about the changes in the amounts and types of these cell envelope components as the bacillus divides during in vitro culture. To shed light on these phenomena, we examined growth-dependent changes over time in major cell envelope components of virulent M.tb by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thin-layer chromatography, mass spectrometry, immunoblotting and flow cytometry. Our studies provide evidence that major mannosylated glycoconjugates on the M.tb cell envelope change as M.tb grows in vitro on the widely used Middlebrook 7H11 agar. In particular, our compositional analyses show that from Day 9 to 28 the amounts of mannose-containing molecules, such as mannose-capped lipoarabinomannan, lipomannan and phosphatidyl-myo-inositol mannosides, change continuously in both the cell envelope and outer cell surface. Along with these changes, mannan levels on the outer cell surface also increase significantly over time. The implications of these differences in terms of how M.tb is grown for studies performed in vitro and in vivo for assessing M.tb-host recognition and establishment of infection are discussed.
Mycobacterial Cell Envelope
In 2016, the World Health Organization reported that one person dies of tuberculosis (TB) every 21 s. A host environment that Mycobacterium tuberculosis (M.tb) finds during its route of infection is the lung mucosa bathing the alveolar space located in the deepest regions of the lungs. We published that human lung mucosa, or alveolar lining fluid (ALF), contains an array of hydrolytic enzymes that can significantly alter the M.tb surface during infection by cleaving off parts of its cell wall. This interaction results in two different outcomes: modifications on the M.tb cell wall surface and release of M.tb cell wall fragments into the environment. Typically, one of the first host immune cells at the site of M.tb infection is the neutrophil. Neutrophils can mount an extracellular and intracellular innate immune response to M.tb during infection. We hypothesized that exposure of neutrophils to ALF-induced M.tb released cell wall fragments would prime neutrophils to control M.tb infection better. Our results show that ALF fragments activate neutrophils leading to an increased production of inflammatory cytokines and oxidative radicals. However, neutrophil exposure to these fragments reduces production of chemoattractants (i.e., interleukin-8), and degranulation, with the subsequent reduction of myeloperoxidase release, and does not induce cytotoxicity. Unexpectedly, these ALF fragment-derived modulations in neutrophil activity do not further, either positively or negatively, contribute to the intracellular control of M.tb growth during infection. However, secreted products from neutrophils primed with ALF fragments are capable of regulating the activity of resting macrophages. These results indicate that ALF-induced M.tb fragments could further contribute to the control of M.tb growth and local killing by resident neutrophils by switching on the total oxidative response and limiting migration of neutrophils to the infection site.
Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis, is a major public health challenge facing the world. During infection, M.tb is deposited in the lung alveolar space where it comes in contact with the lung mucosa, known as alveolar lining fluid (ALF), an environment that M.tb encounters at different stages of the infection and disease. ALF is abundant in homeostatic and antimicrobial hydrolytic enzymes, also known as hydrolases. Here we demonstrate that ALF hydrolases, at their physiological concentrations and upon contact with M.tb, release M.tb cell envelope fragments into the milieu. These released fragments are bioactive, but non-cytotoxic, regulate the function of macrophages, and thus are capable of modulating the immune response contributing to the control of M.tb infection by human macrophages. Specifically, macrophages exposed to fragments derived from the exposure of M.tb to ALF were able to control the infection primarily by increasing phagosome-lysosome fusion and acidification events. This enhanced control was found to be dependent on fragment-induced interleukin-10 (IL-10) production but also involves the STAT3 signaling pathway in an IL-10-independent manner. Collectively our data indicate that M.tb fragments released upon contact with lung mucosa hydrolases participate in the host immune response to M.tb infection through innate immune modulation.
The Mycobacterium tuberculosis cell envelope has been evolving over time to make the bacterium transmissible and adaptable to the human host. In this context, the M. tuberculosis cell envelope contains a peripheral barrier full of lipids, some of them unique, which confer M. tuberculosis with a unique shield against the different host environments that the bacterium will encounter at the different stages of infection. This lipid barrier is mainly composed of glycolipids that can be characterized by three different subsets: trehalose-containing, mannose-containing, and 6-deoxy-pyranose-containing glycolipids. In this review, we explore the roles of these cell envelope glycolipids in M. tuberculosis virulence and pathogenesis, drug resistance, and further, how these glycolipids may dictate the M. tuberculosis cell envelope evolution from ancient to modern strains. Finally, we address how these M. tuberculosis cell envelope glycolipids are impacted by the host lung alveolar environment, their role in vaccination and masking host immunity, and subsequently the impact of these glycolipids in shaping how M. tuberculosis interacts with host cells, manipulating their immune response to favor the establishment of an infection.