Deciphering and Imaging Mechanisms Allowing Fast-Growing Pathogenic Mycobacteria to Acquire a Virulent Invasive Phenotype – DIMYVIR

Two important elements in the smooth to rough comparison appeared when analyzing the transcriptomic data of the S/R transition. First, lsr2 appeared as a gene specifically induced in the rough form as compared to the smooth form. Lsr2 is a pleiotropic regulator potentially involved in the regulation of the glycopeptidolipid biosynthesis, responsible to the smooth aspect of M. abscessus. In addition, a specific genomic region comprises the MAB_4690c (pstA) et 4691c genes which could be involved in the synthesis of an unknown cell wall-associated lipid. We have generated specific mutants of lsr2 and pstA and analyzed their virulence in zebrafish embryos as well as in mice. In addition, a Tn5-based transpositional library in M. abscessus R has been generated. Based on the morphological aspect of the colonies, 7 mutants were selected and analyzed with respect to their capacity to produce biofilms and cords. The same fluorescent mutants were then injected intravenously in zebrafish embryos. Their capacity to colonize lungs was next investigated in the murine model.
Transcriptomic analyses by qRT-PCR, combined with the use of transgenic zebrafish lines and the antisens technology were exploited to determine the contribution of innate immunity and inflammation during infection with M. abscessus. Importantly, a new technological development was achieved to evaluate the bacterial burden during infection in zebrafish. It relies on the determination of fluorescent pixel counts of the fluorescent mycobacteria, which reflects the status of the bacterial loads. On major advantage of this technique is that it does not require to sacrifice the animal, thus allowing to study the dynamic of the infection and bacterial growth in the same individual.

Among the 7 cell wall mutants selected, two were found to be particularly interesting, being more attenuated in zebrafish than the parental R strain. One of them, 1C3 (possessing a Tn5 insertion within Mabs_4024c encoding a putative monooxygenase) has conserved the ability to produce cords, whereas 22D1 with Tn5 insertion within Mabs_3035 and involved in phenazine biosynthesis has lost the capacity to make cords.
Regarding Lsr2, our results indicate that a mutation within lsr2 in M. abscessus R abolishes completely the virulence of the strain, the capacity to produce cords and granulomas in infected zebrafish. With respect to pstA, the corresponding mutant was found to be more attenuated in infected mice as compared to the parental R strain, a phenotype which has also been confirmed in zebrafish.
One major breakthrough of DIMYVIR came from the description for the first time of a mutant in M. abscessus R, devoid of cording and particularly attenuated in two complementary animal models. This already allowed us to confirm our original hypothesis that the capacity to produce cords is highly connected to the virulence of M. abscessus R and opens up to new possibilities of intervention to control this infection.
The analyses combining qRT-PCR and several zebrafish transgenic lines unraveled the high propensity of the R variant to induce a pro-inflammatory response characterized by a strong TNF induction by macrophage and a massive recruitment of neutrophils. The role of TNF has been further confirmed thanks to the use of TNF-receptor morphants, displaying a hypercording phenotype, leading to a rapid development of abscesses and early larval death. These results confirm the importance of the pro-inflammatory response and emphasize the protective role of TNF toward M. abscesus infection.

From a fundamental point of view, DIMYVIR aims to establish new concepts related to regulatory mechanisms in pathogenic mycobacteria and particularly into the physiopathology of M. abscessus which represents the most frequently encountered mycobacterial rapid grower in human infections. In addition, deciphering the molecular bases of the S to R morphotype switch, therefore, promises to reveal new insight into the evolution of environmental mycobacteria towards pathogenicity. Unraveling mechanisms controlling the R morphotype will help to establish a framework for assessing the clinical correlation between M. abscessus infection and colony phenotype. From a clinical perspective, this project will help to open the field to new therapeutic and diagnostic developments by selectively targeting the S to R transition and in predicting the switch in patients to prevent the development of the severe clinical forms. The therapeutic issue appears extremely important, as M. abscesuss is notorious for being the most drug-resistant mycobacterial species, being equipped with a complex hydrophobic cell wall that constitutes an efficient impermeability barrier to most antibiotics and disinfectants. It is naturally resistant to conventional antitubercular drugs, thus severely restricting the number of molecules available for treatment. Therefore, we believe that this project presented will lead to important achievements for a rapid progress towards effective diagnostics and therapeutics against the pathology developed by this emerging pathogen and other related mycobacteria.

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3. A. Bernut, V. Le Moigne, T. Lesne, G. Lutfalla, J-L. Herrmann and L. Kremer. 2014. In vivo assessment of drug efficacy against Mycobacterium abscessus using the embryonic zebrafish test system. Antimicrobial Agents Chemother. 58: 4054-4063.

4. A. Bernut, G. Lutfalla, and L. Kremer. 2015. Regards au travers du Danio pour mieux comprendre les interactions hôtes/pathogènes. Med. Sci. In Press.

Mycobacterium abscessus is currently the most frequently isolated rapid-growing mycobacteria (RGM) in human pathology and the major RGM involved in lung infections. It has recently emerged as responsible for severe pulmonary infections in patients with cystic fibrosis (CF) or those who have undergone lung transplantation. In addition, it represents the most antibiotic resistant mycobacterial species and is generally only sensitive to two antibiotics, clarithromycin and amikacin, thus making treatment extremely difficult.
Key issues of clinical importance are whether isolation of M. abscessus, or other non-tuberculous mycobacteria (NTM), from the sputum of patients represents colonization or invasive pulmonary infection, and whether lung colonization inevitably leads to the development of invasive and persistent infection. NTM have long been recognized as having smooth (S) and rough (R) colony morphology, a phenotype that is influenced by the presence or absence of cell wall associated glycopeptidolipids (GPL), respectively. GPLs are associated with sliding motility and the ability to form biofilms, two characteristics that are thought to play a central role in bacterial colonization, as compared to other biofilm-forming Gram positive and Gram negative bacteria infecting CF patients. In addition, a clear correlation between the R colony morphotype, which produces cords and has increased virulence as compared to the S variant, has also been established. Therefore, the ability of M. abscessus to naturally switch phenotypes may allow the bacteria to transition between a colonizing S microorganism to an R invasive human pathogen. Understanding the molecular bases allowing the transition of a colonizing S variant to a virulent and invasive R variant of M. abscessus is particularly important and may be relevant to other NTM, considering the impact and prevalence of NTM in respiratory diseases like chronic obstructive pulmonary disease (COPD). In this context, this proposal aims to decipher specific mechanisms characterizing the R morphotype and to analyze the in vivo advantages conferred by this transition in infected animals. Our recent transcriptomic/RNAseq studies now gives us access to the whole and precise picture of the transcriptomic comparison between S and R variants of M. abscessus. The role and contribution of several genes identified by the transcriptomic approach or genes selected via screening of “cording”-deficient strains from a transposon library developed in the R morphotype, will be evaluated through the use of defined mutants with respect to i) the mycobacterial cell wall modifications and ii) the virulence in infected mice and zebrafish embryos, both models which are also amenable to mimic a CF-like environment. Additionally, infection with the various mutants will be followed-up at spatial and temporal levels, thanks to the optical transparency of zebrafish embryos. The participation/recruitment of macrophages and neutrophils and incidence of the pro-inflammatory response to infection will also be addressed. Overall, this project will help to unravel and describe specific virulence determinants and/or regulatory pathways related to the mechanisms of invasive and chronic lung infection stage of the disease.
Deciphering the molecular basis of the S to R morphotype switch, therefore, promises to reveal new insight into the evolution of environmental mycobacteria towards pathogenicity. From a clinical perspective, it is also intended to propose innovative drug developments, for instance by selective targeting of the S to R transition, which is particularly warranted due to the extreme resistance of M. abscessus to most current antibiotics.
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