Altogether these data suggested that GlmU

Altogether these data suggested that GlmU might be involved in the M. smegmatis biofilm mediated defence mechanisms. GlmU belongs to a metabolic pathway leading to UDP-GlcNAc from fructose-1-phosphate involved in the biosynthesis of peptidoglycan and lipopolysaccharide (Fig. 8). Recently, we demonstrated that impairment of NanA (N-acetylneuraminate lyase) expression and/or inhibition due to synthetic drugs strongly decrease biofilm formation in E.coli (18). Interestingly, NanA belongs to the same pathway connecting sialic Torin1 metabolism and the amino sugars biosynthesis where GlmU is located, as shown in Fig. 8. Moreover, although the nanA gene is absent in M. smegmatis genome, NanA and GlmU proteins occur within the proteome of many well known pathogenic bacteria such as Staphylococcus aureus (both NanA and GlmU), Klebsiella pneumoniae (GlmU) and Pseudomonas aeruginosa (GlmU). Our findings pointed out to sialic acid metabolism as a key pathway in biofilm production in M. smegmatis. Sialic acid and its metabolites GlcNAc and GlcNAc-6-P have long been recognised as signalling molecules in biofilm formation, colonization and host invasion (54). Moreover, several genes involved in sialic acid metabolism were shown to play a central role in the mechanism of biofilm formation by pathogenic bacteria both in vitro and in vivo 55, 56. We provided evidence that glmU is involved in the biosynthesis and assembly of M. smegmatis biofilm protective architecture. Since the reaction catalyzed by the acetyltransferase domain of GlmU is unique to bacteria not having any homology with eukaryotic organisms, these observations might open up the way to the development of compounds that can effectively target this enzyme specifically hampering biofilm formation and avoiding the risk of generating drug-resistant strains.
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Acknowledgments
Introduction DNA alkylating agents are capable of inducing DNA damage by attaching an alkyl group to DNA, and have been widely used as chemotherapeutic drugs in cancer chemotherapy [1], [2]. On the other hand, PARP-1 is the founding member of the PARP family, a group of nuclear enzymes that play a critical role in DNA damage repair through poly(ADP-ribosyl)ation [3]. It is known that poly(ADP-ribosyl)ation is an energetically expensive process, causing rapid depletion of cellular β-nicotinamide adenine dinucleotide (NAD+), failure in ATP production, and eventually necrotic cell death [4]. It has been reported that N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), a commonly used alkylating agent, kills cells by necrosis through massive production of DNA strand breaks, over-activation of PARP and ATP depletion [5], [6]. Macroautophagy (referred as autophagy hereafter in this study) is a cellular catabolic degradation process in response to starvation or other stress conditions whereby cellular proteins and organelles are engulfed to autophagosomes and eventually digested via lysosomes to sustain cellular homeostasis [7], [8]. Although it is still controversial regarding the role of autophagy in cell death or cell survival, accumulating evidence tends to suggest that autophagy generally serves as a pro-survival mechanisms, especially under stress conditions, such starvation, oxidative stress, metabolic stress and DNA damage [9], [10], [11]. So far, there are limited reports regarding the involvement of autophagy in the response to DNA alkylating agents. An early study showed that MNNG induced obvious increase of autophagic vacuoles in syrian golden hamster pancreatic organ explants [12]. Moreover, MNNG treatment was reported to induce autophagy in Myc/p53ERTAM lymphoma cells in vivo and knockdown of ATG5 significantly sensitized MNNG-induced cytotoxic effect [13], suggesting that autophagy serves as a survival pathway in tumor cells treated with alkylating agents. At present the signaling pathways involved in alkylating agent-induced autophagy are still elusive. In this study we have identified that PARP-1 activation, ATP depletion and subsequent activation of AMP-activated protein kinase (AMPK) play an important role in the induction of autophagy and such autophagy serves as a pro-survival function in MNNG-induced necrotic cell death.