Succinylome Analysis Reveals the Involvement of Lysine Succinylation in Metabolism in Pathogenic Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, remains one of the most prevalent human pathogens and a major cause of mortality worldwide. Metabolic network is a central mediator and defining feature of the pathogenicity of Mtb. Increasing evidence suggests that lysine succinylation dynamically regulates enzymes in carbon metabolism in both bacteria and human cells; however, its extent and function in Mtb remain unexplored. Here, we performed a global succinylome analysis of the virulent Mtb strain H37Rv by using high accuracy nano-LC-MS/MS in combination with the enrichment of succinylated peptides from digested cell lysates and subsequent peptide identification. In total, 1545 lysine succinylation sites on 626 proteins were identified in this pathogen. The identified succinylated proteins are involved in various biological processes and a large proportion of the succinylation sites are present on proteins in the central metabolism pathway. Site-specific mutations showed that succinylation is a negative regulatory modification on the enzymatic activity of acetyl-CoA synthetase. Molecular dynamics simulations demonstrated that succinylation affects the conformational stability of acetyl-CoA synthetase, which is critical for its enzymatic activity. Further functional studies showed that CobB, a sirtuin-like deacetylase in Mtb, functions as a desuccinylase of acetyl-CoA synthetase in in vitro assays. Together, our findings reveal widespread roles for lysine succinylation in regulating metabolism and diverse processes in Mtb. Our data provide a rich resource for functional analyses of lysine succinylation and facilitate the dissection of metabolic networks in this life-threatening pathogen.Post-translational modifications (PTMs)¹ are complex and fundamental mechanisms modulating diverse protein properties and functions, and have been associated with almost all known cellular pathways and disease processes (1, 2). Among the hundreds of different PTMs, acylations at lysine residues, such as acetylation (3–6), malonylation (7, 8), crotonylation (9, 10), propionylation (11–13), butyrylation (11, 13), and succinylation (7, 14–16) are crucial for functional regulations of many prokaryotic and eukaryotic proteins. Because these lysine PTMs depend on the acyl-CoA metabolic intermediates, such as acetyl-CoA (Ac-CoA), succinyl-CoA, and malonyl-CoA, lysine acylation could provide a mechanism to respond to changes in the energy status of the cell and regulate energy metabolism and the key metabolic pathways in diverse organisms (17, 18).Among these lysine PTMs, lysine succinylation is a highly dynamic and regulated PTM defined as transfer of a succinyl group (-CO-CH₂-CH₂-CO-) to a lysine residue of a protein molecule (8). It was recently identified and comprehensively validated in both bacterial and mammalian cells (8, 14, 16). It was also identified in core histones, suggesting that lysine succinylation may regulate the functions of histones and affect chromatin structure and gene expression (7). Accumulating evidence suggests that lysine succinylation is a widespread and important PTM in both eukaryotes and prokaryotes and regulates diverse cellular processes (16). The system-wide studies involving lysine-succinylated peptide immunoprecipitation and liquid chromatography-mass spectrometry (LC-MS/MS) have been employed to analyze the bacteria (E. coli) (14, 16), yeast (S. cerevisiae), human (HeLa) cells, and mouse embryonic fibroblasts and liver cells (16, 19). These succinylome studies have generated large data sets of lysine-succinylated proteins in both eukaryotes and prokaryotes and demonstrated the diverse cellular functions of this PTM. Notably, lysine succinylation is widespread among diverse mitochondrial metabolic enzymes that are involved in fatty acid metabolism, amino acid degradation, and the tricarboxylic acid cycle (19, 20). Thus, lysine succinylation is reported as a functional PTM with the potential to impact mitochondrial metabolism and coordinate different metabolic pathways in human cells and bacteria (14, 19–22).Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a major cause of mortality worldwide and claims more human lives annually than any other bacterial pathogen (23). About one third of the world''s population is infected with Mtb, which leads to nearly 1.3 million deaths and 8.6 million new cases of TB in 2012 worldwide (24). Mtb remains a major threat to global health, especially in the developing countries. Emergence of multidrug resistant (MDR) and extensively drug-resistant (XDR) Mtb, and also the emergence of co-infection between TB and HIV have further worsened the situation (25–27). Among bacterial pathogens, Mtb has a distinctive life cycle spanning different environments and developmental stages (28). Especially, Mtb can exist in dormant or active states in the host, leading to asymptomatic latent TB infection or active TB disease (29). To achieve these different physiologic states, Mtb developed a mechanism to sense diverse signals from the host and to coordinately regulate multiple cellular processes and pathways (30, 31). Mtb has evolved its metabolic network to both maintain and propagate its survival as a species within humans (32–35). It is well accepted that metabolic network is a central mediator and defining feature of the pathogenicity of Mtb (23, 36–38). Knowledge of the regulation of metabolic pathways used by Mtb during infection is therefore important for understanding its pathogenicity, and can also guide the development of novel drug therapies (39). On the other hand, increasing evidence suggests that lysine succinylation dynamically regulates enzymes in carbon metabolism in both bacteria and human cells (14, 19–22). It is tempting to speculate that lysine succinylation may play an important regulatory role in metabolic processes in Mtb. However, to the best of our knowledge, no succinylated protein in Mtb has been identified, presenting a major obstacle to understand the regulatory roles of lysine succinylation in this life-threatening pathogen.In order to fill this gap in our knowledge, we have initiated a systematic study of the identities and functional roles of the succinylated protein in Mtb. Because Mtb H37Rv is the first sequenced Mtb strain (40) and has been extensively used for studies in dissecting the roles of individual genes in pathogenesis (41), it was selected as a test case. We analyzed the succinylome of Mtb H37Rv by using high accuracy nano-LC-MS/MS in combination with the enrichment of succinylated peptides from digested cell lysates and subsequent peptide identification. In total, 1545 lysine succinylation sites on 626 proteins were identified in this pathogen. The identified succinylated proteins are involved in various biological processes and render particular enrichment to metabolic process. A large proportion of the succinylation sites are present on proteins in the central metabolism pathway. We further dissected the regulatory role of succinylation on acetyl-CoA synthetase (Acs) via site-specific mutagenesis analysis and molecular dynamics (MD) simulations showed that reversible lysine succinylation could inhibit the activity of Acs. Further functional studies showed that CobB, a sirtuin-like deacetylase in Mtb, functions as a deacetylase and as a desuccinylase of Acs in in vitro assays. Together, our findings provide significant insights into the range of functions regulated by lysine succinylation in Mtb.     

Proteomic Identification of Novel Secreted Antibacterial Toxins of the Serratia marcescens Type VI Secretion System

It has recently become apparent that the Type VI secretion system (T6SS) is a complex macromolecular machine used by many bacterial species to inject effector proteins into eukaryotic or bacterial cells, with significant implications for virulence and interbacterial competition. “Antibacterial” T6SSs, such as the one elaborated by the opportunistic human pathogen, Serratia marcescens, confer on the secreting bacterium the ability to rapidly and efficiently kill rival bacteria. Identification of secreted substrates of the T6SS is critical to understanding its role and ability to kill other cells, but only a limited number of effectors have been reported so far. Here we report the successful use of label-free quantitative mass spectrometry to identify at least eleven substrates of the S. marcescens T6SS, including four novel effector proteins which are distinct from other T6SS-secreted proteins reported to date. These new effectors were confirmed as antibacterial toxins and self-protecting immunity proteins able to neutralize their cognate toxins were identified. The global secretomic study also unexpectedly revealed that protein phosphorylation-based post-translational regulation of the S. marcescens T6SS differs from that of the paradigm, H1-T6SS of Pseudomonas aeruginosa. Combined phosphoproteomic and genetic analyses demonstrated that conserved PpkA-dependent threonine phosphorylation of the T6SS structural component Fha is required for T6SS activation in S. marcescens and that the phosphatase PppA can reverse this modification. However, the signal and mechanism of PpkA activation is distinct from that observed previously and does not appear to require cell–cell contact. Hence this study has not only demonstrated that new and species-specific portfolios of antibacterial effectors are secreted by the T6SS, but also shown for the first time that PpkA-dependent post-translational regulation of the T6SS is tailored to fit the needs of different bacterial species.Gram-negative bacteria have evolved several specialized protein secretion systems to secrete a wide variety of substrate proteins into the extracellular milieu or to inject them into other, often eukaryotic, cells (1). Secreted proteins and their associated secretion systems are very important in bacterial virulence and interactions with other organisms (2). One of the most recent discoveries in this field is the Type VI secretion system (T6SS),¹ which occurs widely across bacterial species (3, 4) and can target proteins to both bacterial and eukaryotic cells (5). The significance of the T6SS is becoming increasingly apparent. It has been implicated in virulence, commensalism, and symbiosis with eukaryotes (5, 6). Additionally, in many bacteria, the T6SS is now implicated in antibacterial activity. T6SS-mediated antibacterial killing appears to be important for competition between bacterial species, for example within the resident microflora of a eukaryotic host (5, 7).Secretion by the T6SS relies on 13 conserved core components which are predicted to form a large machinery associated with the cell envelope, including membrane-bound and bacteriophage tail-like subassemblies (8, 9). The membrane bound subassembly consists of inner membrane proteins (TssLM) and an outer membrane lipoprotein (TssJ) and is anchored to the cell wall. The phage tail-like assembly consists of several proteins that show structural homology with T4 phage tail proteins or are organized in similar structures (10). Hcp (TssD) proteins form hexameric rings and are thought to stack into tube-like structures (11, 12). This Hcp tube is believed to be capped by a trimer of VgrG (TssI) proteins, which share structural homology with the needle of the T4 phage tail (10, 13). In addition, VipA (TssB) and VipB (TssC) form a large tubular structure highly reminiscent of the T4 phage tail sheath (14, 15). Such similarities have led to the idea that the T6SS resembles an inverted contractile bacteriophage infection machinery and injects substrates via an Hcp/VgrG needle into other cells. Recent models propose that the VipA/B sheath surrounds the Hcp/VgrG needle and contraction of the VipA/B tube pushes the Hcp/VgrG needle out of the cell (16–18). It has been postulated that this mechanism can be triggered by close contact with other neighboring cells (19–21).Assembly, localization, and remodelling of VipA/B tubules in vivo depend on the AAA+ ATPase ClpV (TssH), another essential core component of the T6SS (14, 16, 17). ClpV also interacts with the accessory component Fha (TagH) (22, 23), which is found in a subset of T6SSs (4). The Fha protein has an N-terminal domain with a forkhead associated motif, which is predicted to bind phospho-threonine peptides (24). In Pseudomonas aeruginosa, Fha1 is phosphorylated by the Thr/Ser kinase PpkA (TagE) and dephosphorylated by the phosphatase PppA (TagG), and the phosphorylation state of Fha1 regulates the activity of the T6SS (22, 23). Phosphorylation of Fha in P. aeruginosa is also controlled by additional components, which act upstream of PpkA and form a regulatory cascade for T6SS activation (22, 25). Although homologs of PpkA and PppA have been identified in the T6SS gene clusters of certain other bacteria (3), the regulation of the T6SS by post-translational protein phosphorylation has not yet been experimentally investigated outside of Pseudomonas.To understand how the T6SS affects eukaryotic and bacterial cells, it is critical to identify substrate proteins secreted by the T6SS. The VgrG and Hcp proteins were the first identified T6SS substrates and appear to be generally secreted to the external milieu by all T6SSs (26). However, as mentioned above, Hcp and VgrG are core components of the T6SS machinery and therefore represent extracellular components of the secretion apparatus rather than genuine secreted effector proteins. Nonetheless, a limited number of VgrG homologs with extra functional effector domains at the C terminus have been identified or predicted, which account for some of the T6SS dependent effects seen against bacteria and eukaryotes. For example, the C-terminal domain of VgrG-1 from Vibrio cholerae shows actin crosslinking activity in eukaryotic cells (13, 27) and the C-terminal domain of V. cholerae VgrG-3 has bacterial cell wall hydrolase activity (28, 29).Recently, following much effort in the field, a small number of proteins secreted by the T6SS, but not structural components, have been experimentally identified. These proteins are regarded as true secreted substrates of the T6SS, with effector functions in target cells (29–35). For example, antibacterial T6SS-secreted effector proteins with peptidoglycan amidase (cell wall hydrolysis) function, the Type VI amidase effector (Tae) proteins, have been identified in Burkholderia thailandensis (32), P. aeruginosa (31), and Serratia marcescens (30). These Tae proteins play a role in T6SS-mediated antibacterial killing activity and genes encoding four families of Tae protein have been widely identified in other bacteria with T6SSs (32). T6SS-secreted effector proteins which are not peptidoglycan hydrolases have also been reported, including Tse2 secreted by P. aeruginosa, which acts in the bacterial cytoplasm (31), and the VasX and TseL proteins secreted by the V. cholerae T6SS, which are suggested to target membrane lipids (29, 34, 35). In the case of antibacterial T6SSs, the secreting bacterial cells are protected from their own T6SS effector proteins by specific immunity proteins (29–32, 35). However, given the large number of T6SSs in different bacterial species and their apparent ability to secrete multiple substrates, experimentally identified T6-secreted effector proteins still remain surprisingly scarce.Here we report the identification of multiple T6SS-secreted effector proteins in S. marcescens. S. marcescens is an opportunistic pathogen, for example causing ocular infections, nosocomial septicemia and pneumonia (36). Previously, we have identified a T6SS in S. marcescens Db10, which targets and efficiently kills other bacterial cells and plays a role in antibacterial competition (37). We have recently demonstrated that this T6SS secretes two antibacterial effectors, the Tae4 homologs Ssp1 and Ssp2, with cognate immunity proteins Rap1a and Rap2a (30).In this work, we report the analysis of the T6SS-dependent secretome of S. marcescens by label-free quantitation (LFQ) mass spectrometry and describe the identification and characterization of four novel T6SS-secreted effector proteins. These were confirmed as antibacterial toxins and specific immunity proteins were identified. Additionally, this global secretomic analysis, in combination with genetic and phosphoproteomic analyses, demonstrated that a post-translational phosphorylation system influences the ability of the S. marcescens T6SS to secrete effector proteins. Although this system uses homologs of the P. aeruginosa PpkA, PppA and Fha components, the circumstances and impact of Fha phosphorylation were shown to vary between organisms.     

Calcium Is Essential for the Major Pseudopilin in the Type 2 Secretion System

The pseudopilus is a key feature of the type 2 secretion system (T2SS) and is made up of multiple pseudopilins that are similar in fold to the type 4 pilins. However, pilins have disulfide bridges, whereas the major pseudopilins of T2SS do not. A key question is therefore how the pseudopilins, and in particular, the most abundant major pseudopilin, GspG, obtain sufficient stability to perform their function. Crystal structures of Vibrio cholerae, Vibrio vulnificus, and enterohemorrhagic Escherichia coli (EHEC) GspG were elucidated, and all show a calcium ion bound at the same site. Conservation of the calcium ligands fully supports the suggestion that calcium ion binding by the major pseudopilin is essential for the T2SS. Functional studies of GspG with mutated calcium ion-coordinating ligands were performed to investigate this hypothesis and show that in vivo protease secretion by the T2SS is severely impaired. Taking all evidence together, this allows the conclusion that, in complete contrast to the situation in the type 4 pili system homologs, in the T2SS, the major protein component of the central pseudopilus is dependent on calcium ions for activity.In Gram-negative bacteria, the type 2 secretion system (T2SS)² is used for the secretion of several important proteins across the outer membrane (1). The T2SS is also called the terminal branch of the general secretory pathway (Gsp) (2) and, in Vibrio species, the extracellular protein secretion (Eps) apparatus (3). This sophisticated multiprotein machinery spans both the inner and the outer membrane of Gram-negative bacteria and contains 11–15 different proteins. The T2SS consists of three major subassemblies (4–9): (i) the outer membrane complex comprising mainly the crucial multisubunit secretin GspD; (ii) the pseudopilus, which consists of one major and several minor pseudopilins; and (iii) an inner membrane platform, containing the cytoplasmic secretion ATPase GspE and the membrane proteins GspL, GspM, GspC, and GspF.The pseudopilus is a key element of the T2SS that forms a helical fiber spanning the periplasm. The fiber is assembled from multiple subunits of the major pseudopilin GspG (4, 5, 10–14). The pseudopilus is thought to form a plug of the secretin pore in the outer membrane and/or to function as a piston during protein secretion. In recent years, studies of the T2SS pseudopilins led to structure determinations of all individual pseudopilins (13, 15–17). The recent structure of the helical ternary complex of GspK-GspI-GspJ suggested that these three minor pseudopilins form the tip of the pseudopilus (17). A crystal structure of GspG from Klebsiella oxytoca was in a previous study combined with electron microscopy data to arrive at a helical arrangement, with no evidence for special features, such as disulfide bridges, other covalent links, or metal-binding sites, for stabilizing this major pseudopilin or the pseudopilus (13).The pseudopilins of the T2SS share a common fold with the type 4 pilins (15–21). Pilins are proteins incorporated into pili, long appendages on the surface of bacteria forming thin, strong fibers with multiple functions (19, 21). Type 4 pilins and pseudopilins contain a prepilin leader sequence that is cleaved off by a prepilin peptidase, yielding mature protein (10, 11, 22). A distinct feature of the type 4 pilins is the occurrence of a disulfide bridge connecting β4 to a Cys in the so-called “D-region” near the C terminus (21). In a recent study (23) on the thin fibers of Gram-positive bacteria, isopeptide units appeared to be essential for providing these filaments sufficient cohesion and stability. A key question was therefore whether the major pseudopilin GspG also requires a special feature to obtain sufficient stability to perform its function.