A new Mycobacterium tuberculosis smooth colony reduces growth inside human macrophages and represses PDIM Operon gene expression. Does an heterogeneous population exist in intracellular mycobacteria?

Abstract

Mycobacterium tuberculosis (MTB) colony morphology was associated to the pathogen's virulence. We isolated a new MTB H37Rv smooth colony, which only appeared following human macrophages (MDM) infection. The new phenotype was Alcohol-Acid resistant, but devoid of a covering capsule and biofilm defective. We ascertained that there were no deletions in the Rv0096-Rv0101 PDIM Operon, but that its expression was repressed as compared to MTB wild type (wt). Its lipid composition displayed lower PDIM components and higher TAG as compared to wt. In MDM it induced the sigma factors sigB, sigI and sigL expression vs. synthetic medium culture, while it repressed other six sigma factors. It also induced, significantly more than wt, mprA, a mycobacterial persistence regulator.

It was phagocytosed more than wt by MDM, where it grew significantly less, but persisted therein till 14 days infection. It induced significantly less IFN-γ, IL-12 and IL-27 transcription than wt in infected MDM, while it increased the transcription of inducible NOS. It resided in mature, LAMP-3 positive phagolysosomes, where it never formed cords. This apparently “weaker” colony might represent an adaptive intracellular phenotype, whose infection may be less productive, but probably better equipped for a long lasting persistence in mildly activated host cells.

Introduction

Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis (TB) and is one of the most important human pathogens. The incidence of tuberculosis continues to increase worldwide, and it has been estimated that one-third of the world's population is latently infected with MTB [1].

Despite decades of intense investigation, the nature of virulence determinants and the molecular mechanisms of M. tuberculosis pathogenesis are still largely unknown [2], [3].

Little is known about the population structure of different mycobacterial strains, and the contribution of important characteristic such us morphology, virulence and adaptation, on disease progression.

Recent genetic analyses have identified specific lipids of the cell envelope as important effectors in MTB pathogenesis [4]. An interesting feature of the mycobacteria envelope is its high lipid content, among these the phthiocerol dimycocerosates (PDIM) has been studied intensively and shown to promote Mtb virulence [5]. In this respect, it has been recently identified a new Operon (Rv0096-Rv0101) involved in PDIM biosynthesis and showed that a mutation introduced in this Operon gave rise to a new phenotype of the Mycobacterium bovis-BCG colony [6].

The complex, lipid-rich, and hydrophobic structure is responsible for the acid-fast staining properties, the distinctive colony morphology, the biofilm formation, and the innate antibiotic resistance of MTB [7]. Defects in cell wall components, and outermost covering material, can modify the surface properties of individual cells, alter cell-to-cell interactions, and ultimately give rise to colonies with different morphologies [8].

When grown on solid agar, virulent MTB tend to form colonies with irregular spreading margins and highly textured surfaces (rough phenotype), while the cultures of the attenuated MTB typically grow as colonies with regular smooth surfaces. Several studies demonstrated that the virulence of an individual mycobacterial strain could be predicted by quantization of the strength of its cording (serpentine cords) [9]. In different mycobacterial species smooth variants of the classical rough colony have been found, which usually display a less virulent phenotype in murine models, are biofilm defective and lack different lipids which are, instead, always found in their parental strains [10]. Recently it has been shown that the phenotypic differences of MTB colonies raised during in vitro passages, can reflect the plasticity of MTB genome, due to nucleotide substitutions and insertion/deletions, multiple IS6110 transposition events [11].

In Staphylococcus aureus there are strains capable to produce subpopulations which are phenotypically very different from the parental strain [12]. These naturally occurring subpopulations, named small colony variants (SCV) are better able to persist in mammalian cells and are less susceptible to antibiotics than their wild-type counterparts, and can cause latent or recurrent infections on emergence from the protective environment of the host cell [13].

In this study we describe a new H37Rv colony smooth phenotype, which appeared only after infection of human Monocyte-derived-Macrophages (MDM), while it was never found in synthetic medium culture.

This colony exhibits the typical pattern of the attenuated microorganisms [10] in that it shows a smooth surface, with a fractal-like shaped border when grown on solid media, and, when grown in liquid media, it does not make biofilm on the medium surface, and it never forms serpentine cords.

We compared the ability of the MTB colony new phenotype to invade and survive within MDM with that of wt phenotype, and, contemporarily, we analyzed the MDM response to the infection with the two different colonies in terms of gene expression of inflammatory cytokines.

The potential role of the newly identified colony in persistence in the host's cells and the hypotheses that also in M. tuberculosis species it might arise a bistable and heterogeneous bacterial population [14] is discussed.