Non-Tuberculous Mycobacteria: Molecular and Physiological Bases of Virulence and Adaptation to Ecological Niches

Abstract

Non-tuberculous mycobacteria (NTM) are paradigmatic colonizers of the total environment, circulating at the interfaces of the atmosphere, lithosphere, hydrosphere, biosphere, and anthroposphere. Their striking adaptive ecology on the interconnection of multiple spheres results from the combination of several biological features related to their exclusive hydrophobic and lipid-rich impermeable cell wall, transcriptional regulation signatures, biofilm phenotype, and symbiosis with protozoa. This unique blend of traits is reviewed in this work, with highlights to the prodigious plasticity and persistence hallmarks of NTM in a wide diversity of environments, from extreme natural milieus to microniches in the human body. Knowledge on the taxonomy, evolution, and functional diversity of NTM is updated, as well as the molecular and physiological bases for environmental adaptation, tolerance to xenobiotics, and infection biology in the human and non-human host. The complex interplay between individual, species-specific and ecological niche traits contributing to NTM resilience across ecosystems are also explored. This work hinges current understandings of NTM, approaching their biology and heterogeneity from several angles and reinforcing the complexity of these microorganisms often associated with a multiplicity of diseases, including pulmonary, soft-tissue, or milliary. In addition to emphasizing the cornerstones of knowledge involving these bacteria, we identify research gaps that need to be addressed, stressing out the need for decision-makers to recognize NTM infection as a public health issue that has to be tackled, especially when considering an increasingly susceptible elderly and immunocompromised population in developed countries, as well as in low- or middle-income countries, where NTM infections are still highly misdiagnosed and neglected.

Keywords: environmental mycobacteria; mycobacterial diagnostics; mycobacterial ecology; mycobacterial infection; mycobacterial physiology; non-tuberculous mycobacteria.

Figure 1

Evolution of the short helix 18 in the region B of the 16S rRNA gene. A 12-nucleotide insertion differentiates rapid-growing mycobacteria from slow-growing mycobacteria, except for M. simiae complex that shows an ancestral-like short helix 18 sequence and M. terrae complex that possesses a 14-nucleotide insertion, instead of the typical 12-nucleotide insertion.

Figure 2

The resilient biology of non-tuberculous mycobacteria. (A) Non-tuberculous mycobacteria (NTM) possess a unique outer membrane with several biolayers that increase cell hydrophobicity and environmental resistance. In particular, several classes of lipids are extremely important for mycobacteria sliding motility and cell attachment to the surface, triggering early biofilm formation. (B) Biofilm formation is quorum-sensing dependent with oxidative stress (ROS/NOS) and divalent cations (A²⁺) promoting whiB3 and luxR gene expression, with the consequent increase production of autoinducer-2 (AI-2), leading to the increase in cyclic diguanylate (c-di-GMP) activation and biofilm formation-associated gene expression. Contrary, humic acids inhibit gene expression of biofilm formation-related genes. Additionally, eDNA is secreted into the extracellular matrix by FtsK/SpoIIIE secretion system. (C) NTM are also resistant to recalcitrant compounds, such as polycyclic aromatic hydrocarbons (PAH) and heavy metals. The degradation of PAHs is achieved by a dioxygenase system composed of a dehydrogenase, the dioxygenase small (beta)-subunit, and the dioxygenase large (alpha)-subunit, while mercury (Hg²⁺) triggers the synthesis of mercuric reductases and copper (Cu²⁺) and cadmium (Cd²⁺) are chelated into the mycobacterial cell wall as sulfides. (D) NTM can resist antimicrobials by several mechanisms, including: the intrinsic cell wall provides a physical barrier towards the entrance of antimicrobials; the erm genes that cause the methylation of rRNA, resulting in resistance to macrolides, lincosamide, and streptogramin B; and the expression of efflux pumps of different superfamilies, namely multidrug and toxic compound extrusion (MATE), resistance-nodulation-cell division (RND), ATP-binding cassette (ABC), major facilitator (MFS), and small multidrug resistance (SMR), that actively secrete several antimicrobial compounds. The information present in this figure was combined from data reported by several studies regarding various non-tuberculous mycobacteria species.

Figure 3

Biofilm formation of Mycobacterium smegmatis. M. smegmatis cells can be present in suspension on the environment (I), but under several environmental conditions, they aggregate in microcolonies (II), followed by an increase in cell density and formation of an extracellular matrix (III), leading to the formation of a mature biofilm (IV). In M. smegmatis, biofilm formation is iron (Fe³⁺)-dependent, producing siderophores (e.g., exochelin) responsible for chelating and transporting iron into the mycobacterial cell wall transporters (e.g., iron ATP-binding cassette (ABC) transporters or mycobactin). Monomeromycolyl-diacylglycerol (MMDAG) are important cell wall components involved in biofilm formation, with their secretion being dependent of Lsr2 and GroEL1 proteins regulation of the KasA protein synthesis and MMDAG secretion by mmpl11 efflux pump. Additionally, the Rv0024 gene promotes biofilm formation, while the glmM gene inhibits it. Mycolic acids suffer a transformation from long mycolic acids (C₇₀–C₉₀) into short mycolic acids (C₅₆–C₆₈) when mycobacteria enter the biofilm state. The formation of those short mycolic acids is regulated by the ability of KasA to synthesize trehalose dimycolate, the ability of serine esterase to hydrolyze trehalose dimycolate into short mycolic acids, and by the mce1 operon. Additionally, short mycolic acids are secreted into the extracellular matrix. Mycobacteria cells in the biofilm phenotype have a reinforcing capacity to survive oxidative stress by expressing the redox homeostatic system (RHOCS). In this system, increasing concentration of intracellular NADH promotes the synthesis of PknG protein, which phosphorylates the L13 protein, increasing the binding capacity of L13 with RenU, leading to the oxidation of NADH into NAD⁺ and H⁺.

Figure 4

Mycobacteria–protozoa symbiosis. Pathogenicity islands (PI) promote mycobacteria invasion into amoeba, leading to phagocytosis. Three outcomes can result from this phagocytosis process: mycobacteria evade the lysosome and multiply, causing amoeba lysis; mycobacteria cannot evade the lysosome, causing mycobacteria death; or a symbiotic relationship occurs, with phosphatases encoded by the ptpA, ptpB, sapM genes being produced and excreted by the ESX-4 type VII secretion system. The symbiosis can lead to mycobacteria encystment or escapement from the amoeba. The information presented in this figure was combined from several studies regarding various non-tuberculous mycobacteria species.