Full activation of Enterococcus faecalis gelatinase by a C-terminal proteolytic cleavage

Enterococci account for nearly 10% of all nosocomial infections and constitute a significant treatment challenge due to their multidrug resistance properties. One of the well-studied virulence factors of Enterococcus faecalis is a secreted bacterial protease, termed gelatinase, which has been shown to contribute to the process of biofilm formation. Gelatinase belongs to the M4 family of bacterial zinc metalloendopeptidases, typified by thermolysin. Gelatinase is synthesized as a preproenzyme consisting of a signal sequence, a putative propeptide, and then the mature enzyme. We determined that the molecular mass of the mature protein isolated from culture supernatant was 33,030 Da, which differed from the predicted molecular mass, 34,570 Da, by over 1,500 Da. Using N-terminal sequencing, we confirmed that the mature protein begins at the previously identified sequence VGSEV, thus suggesting that the 1,500-Da molecular mass difference resulted from a C-terminal processing event. By using mutants with site-directed mutations within a predicted C-terminal processing site and mutants with C-terminal deletions fused to a hexahistidine tag, we determined that the processing site is likely to be between residues D304 and 1305 and that it requires the Q306 residue. The results suggest that the E. faecalis gelatinase requires C-terminal processing for full activation of protease activity, making it a unique enzyme among the members of the M4 family of proteases of gram-positive bacteria.Instituto de Biotecnologia y Biologia Molecula

Full activation of Enterococcus faecalis gelatinase by a C-terminal proteolytic cleavage

Enterococci account for nearly 10% of all nosocomial infections and constitute a significant treatment challenge due to their multidrug resistance properties. One of the well-studied virulence factors of Enterococcus faecalis is a secreted bacterial protease, termed gelatinase, which has been shown to contribute to the process of biofilm formation. Gelatinase belongs to the M4 family of bacterial zinc metalloendopeptidases, typified by thermolysin. Gelatinase is synthesized as a preproenzyme consisting of a signal sequence, a putative propeptide, and then the mature enzyme. We determined that the molecular mass of the mature protein isolated from culture supernatant was 33,030 Da, which differed from the predicted molecular mass, 34,570 Da, by over 1,500 Da. Using N-terminal sequencing, we confirmed that the mature protein begins at the previously identified sequence VGSEV, thus suggesting that the 1,500-Da molecular mass difference resulted from a C-terminal processing event. By using mutants with site-directed mutations within a predicted C-terminal processing site and mutants with C-terminal deletions fused to a hexahistidine tag, we determined that the processing site is likely to be between residues D304 and 1305 and that it requires the Q306 residue. The results suggest that the E. faecalis gelatinase requires C-terminal processing for full activation of protease activity, making it a unique enzyme among the members of the M4 family of proteases of gram-positive bacteria.Instituto de Biotecnologia y Biologia Molecula

An integrated computational and experimental study to investigate \u3ci\u3eStaphylococcus aureus\u3c/i\u3e metabolism

Staphylococcus aureus is a metabolically versatile pathogen that colonizes nearly all organs of the human body. A detailed and comprehensive knowledge of staphylococcal metabolism is essential to understand its pathogenesis. To this end, we have reconstructed and experimentally validated an updated and enhanced genome-scale metabolic model of S. aureus USA300_FPR3757. The model combined genome annotation data, reaction stoichiometry, and regulation information from biochemical databases and previous strain-specific models. Reactions in the model were checked and fixed to ensure chemical balance and thermodynamic consistency. To further refine the model, growth assessment of 1920 nonessential mutants from the Nebraska Transposon Mutant Library was performed, and metabolite excretion profiles of important mutants in carbon and nitrogen metabolism were determined. The growth and no-growth inconsistencies between the model predictions and in vivo essentiality data were resolved using extensive manual curation based on optimization-based reconciliation algorithms. Upon intensive curation and refinements, the model contains 863 metabolic genes, 1379 metabolites (including 1159 unique metabolites), and 1545 reactions including transport and exchange reactions. To improve the accuracy and predictability of the model to environmental changes, condition-specific regulation information curated from the existing knowledgebase was incorporated. These critical additions improved the model performance significantly in capturing gene essentiality, substrate utilization, and metabolite production capabilities and increased the ability to generate model-based discoveries of therapeutic significance. Use of this highly curated model will enhance the functional utility of omics data, and therefore, serve as a resource to support future investigations of S. aureus and to augment staphylococcal research worldwide

Urease is an essential component of the acid response network of \u3ci\u3eStaphylococcus\u3c/i\u3e aureus and is required for a persistent murine kidney infection

Staphylococcus aureus causes acute and chronic infections resulting in significant morbidity. Urease, an enzyme that generates NH3 and CO2 from urea, is key to pH homeostasis in bacterial pathogens under acidic stress and nitrogen limitation. However, the function of urease in S. aureus niche colonization and nitrogen metabolism has not been extensively studied. We discovered that urease is essential for pH homeostasis and viability in urea-rich environments under weak acid stress. The regulation of urease transcription by CcpA, Agr, and CodY was identified in this study, implying a complex network that controls urease expression in response to changes in metabolic flux. In addition, it was determined that the endogenous urea derived from arginine is not a significant contributor to the intracellular nitrogen pool in non-acidic conditions. Furthermore, we found that during a murine chronic renal infection, urease facilitates S. aureus persistence by promoting bacterial fitness in the low-pH, urea-rich kidney. Overall, our study establishes that urease in S. aureus is not only a primary component of the acid response network but also an important factor required for persistent murine renal infections

Inactivation of the Pta-AckA Pathway Causes Cell Death in \u3ci\u3eStaphylococcus aureus\u3c/i\u3e

During growth under conditions of glucose and oxygen excess, Staphylococcus aureus predominantly accumulates acetate in the culture medium, suggesting that the phosphotransacetylase-acetate kinase (Pta-AckA) pathway plays a crucial role in bacterial fitness. Previous studies demonstrated that these conditions also induce the S. aureus CidR regulon involved in the control of cell death. Interestingly, the CidR regulon is comprised of only two operons, both encoding pyruvate catabolic enzymes, suggesting an intimate relationship between pyruvate metabolism and cell death. To examine this relationship, we introduced ackA and pta mutations in S. aureus and tested their effects on bacterial growth, carbon and energy metabolism, cid expression, and cell death. Inactivation of the Pta-AckA pathway showed a drastic inhibitory effect on growth and caused accumulation of dead cells in both pta and ackA mutants. Surprisingly, inactivation of the Pta-AckA pathway did not lead to a decrease in the energy status of bacteria, as the intracellular concentrations of ATP, NAD+, and NADH were higher in the mutants. However, inactivation of this pathway increased the rate of glucose consumption, led to a metabolic block at the pyruvate node, and enhanced carbon flux through both glycolysis and the tricarboxylic acid (TCA) cycle. Intriguingly, disruption of the Pta-AckA pathway also induced the CidR regulon, suggesting that activation of alternative pyruvate catabolic pathways could be an important survival strategy for the mutants. Collectively, the results of this study demonstrate the indispensable role of the Pta-AckA pathway in S. aureus for maintaining energy and metabolic homeostasis during overflow metabolism

Staphylococcus aureus ATP Synthase Promotes Biofilm Persistence by Influencing Innate Immunity

ABSTRACT Staphylococcus aureus is a major cause of prosthetic joint infection (PJI), which is characterized by biofilm formation. S. aureus biofilm skews the host immune response toward an anti-inflammatory profile by the increased recruitment of myeloid-derived suppressor cells (MDSCs) that attenuate macrophage proinflammatory activity, leading to chronic infection. A screen of the Nebraska Transposon Mutant Library identified several hits in the ATP synthase operon that elicited a heightened inflammatory response in macrophages and MDSCs, including atpA, which encodes the alpha subunit of ATP synthase. An atpA transposon mutant (ΔatpA) had altered growth kinetics under both planktonic and biofilm conditions, along with a diffuse biofilm architecture that was permissive for leukocyte infiltration, as observed by confocal laser scanning microscopy. Coculture of MDSCs and macrophages with ΔatpA biofilm elicited significant increases in the proinflammatory cytokines interleukin 12p70 (IL-12p70), tumor necrosis factor alpha (TNF-α), and IL-6. This was attributed to increased leukocyte survival resulting from less toxin and protease production by ΔatpA biofilm as determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The enhanced inflammatory response elicited by ΔatpA biofilm was cell lysis-dependent since it was negated by polyanethole sodium sulfanate treatment or deletion of the major autolysin, Atl. In a mouse model of PJI, ΔatpA-infected mice had decreased MDSCs concomitant with increased monocyte/macrophage infiltrates and proinflammatory cytokine production, which resulted in biofilm clearance. These studies identify S. aureus ATP synthase as an important factor in influencing the immune response during biofilm-associated infection and bacterial persistence. IMPORTANCE Medical device-associated biofilm infections are a therapeutic challenge based on their antibiotic tolerance and ability to evade immune-mediated clearance. The virulence determinants responsible for bacterial biofilm to induce a maladaptive immune response remain largely unknown. This study identified a critical role for S. aureus ATP synthase in influencing the host immune response to biofilm infection. An S. aureus ATP synthase alpha subunit mutant (ΔatpA) elicited heightened proinflammatory cytokine production by leukocytes in vitro and in vivo, which coincided with improved biofilm clearance in a mouse model of prosthetic joint infection. The ability of S. aureus ΔatpA to augment host proinflammatory responses was cell lysis-dependent, as inhibition of bacterial lysis by polyanethole sodium sulfanate or a ΔatpAΔatl biofilm did not elicit heightened cytokine production. These studies reveal a critical role for AtpA in shaping the host immune response to S. aureus biofilm

A central role for carbon-overflow pathways in the modulation of bacterial cell death.

Similar to developmental programs in eukaryotes, the death of a subpopulation of cells is thought to benefit bacterial biofilm development. However mechanisms that mediate a tight control over cell death are not clearly understood at the population level. Here we reveal that CidR dependent pyruvate oxidase (CidC) and α-acetolactate synthase/decarboxylase (AlsSD) overflow metabolic pathways, which are active during staphylococcal biofilm development, modulate cell death to achieve optimal biofilm biomass. Whereas acetate derived from CidC activity potentiates cell death in cells by a mechanism dependent on intracellular acidification and respiratory inhibition, AlsSD activity effectively counters CidC action by diverting carbon flux towards neutral rather than acidic byproducts and consuming intracellular protons in the process. Furthermore, the physiological features that accompany metabolic activation of cell death bears remarkable similarities to hallmarks of eukaryotic programmed cell death, including the generation of reactive oxygen species and DNA damage. Finally, we demonstrate that the metabolic modulation of cell death not only affects biofilm development but also biofilm-dependent disease outcomes. Given the ubiquity of such carbon overflow pathways in diverse bacterial species, we propose that the metabolic control of cell death may be a fundamental feature of prokaryotic development

The Staphylococcus aureus CidA and LrgA Proteins Are Functional Holins Involved in the Transport of By-Products of Carbohydrate Metabolism

The Staphylococcus aureus cidABC and lrgAB operons encode members of a well-conserved family of proteins thought to be involved in programmed cell death (PCD). Based on the structural similarities that CidA and LrgA share with bacteriophage holins, we have hypothesized that these proteins function by forming pores within the cytoplasmic membrane. To test this, we utilized a lysis cassette system that demonstrated the abilities of the cidA and lrgA genes to support bacteriophage endolysin-induced cell lysis. Typical of holins, CidA- and LrgA-induced lysis was dependent on the coexpression of endolysin, consistent with the proposed holin-like functions of these proteins. In addition, the CidA and LrgA proteins were shown to localize to the surface of membrane vesicles and cause leakage of small molecules, providing direct evidence of their hole-forming potential. Consistent with recent reports demonstrating a role for the lrgAB homologues in other bacterial and plant species in the transport of by-products of carbohydrate metabolism, we also show that lrgAB is important for S. aureus to utilize pyruvate during microaerobic and anaerobic growth, by promoting the uptake of pyruvate under these conditions. Combined, these data reveal that the CidA and LrgA membrane proteins possess holin-like properties that play an important role in the transport of small by-products of carbohydrate metabolism. IMPORTANCE The Staphylococcus aureus cidABC and lrgAB operons represent the founding members of a large, highly conserved family of genes that span multiple kingdoms of life. Despite the fact that they have been shown to be involved in bacterial PCD, very little is known about the molecular/biochemical functions of the proteins they encode. The results presented in this study reveal that the cidA and lrgA genes encode proteins with bacteriophage holin-like functions, consistent with their roles in cell death. However, these studies also demonstrate that these operons are involved in the transport of small metabolic by-products of carbohydrate metabolism, suggesting an intriguing link between these two seemingly disparate processes

Accumulation of Succinyl Coenzyme A Perturbs the Methicillin-Resistant Staphylococcus aureus (MRSA) Succinylome and Is Associated with Increased Susceptibility to Beta-Lactam Antibiotics

Penicillin binding protein 2a (PBP2a)-dependent resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is regulated by the activity of the tricarboxylic acid (TCA) cycle via a poorly understood mechanism. We report that mutations in sucC and sucD, but not other TCA cycle enzymes, negatively impact β-lactam resistance without changing PBP2a expression. Increased intracellular levels of succinyl coenzyme A (succinyl-CoA) in the sucC mutant significantly perturbed lysine succinylation in the MRSA proteome. Suppressor mutations in sucA or sucB, responsible for succinyl-CoA biosynthesis, reversed sucC mutant phenotypes. The major autolysin (Atl) was the most succinylated protein in the proteome, and increased Atl succinylation in the sucC mutant was associated with loss of autolytic activity. Although PBP2a and PBP2 were also among the most succinylated proteins in the MRSA proteome, peptidoglycan architecture and cross-linking were unchanged in the sucC mutant. These data reveal that perturbation of the MRSA succinylome impacts two interconnected cell wall phenotypes, leading to repression of autolytic activity and increased susceptibility to β-lactam antibiotics. IMPORTANCE mecA-dependent methicillin resistance in MRSA is subject to regulation by numerous accessory factors involved in cell wall biosynthesis, nucleotide signaling, and central metabolism. Here, we report that mutations in the TCA cycle gene, sucC, increased susceptibility to β-lactam antibiotics and was accompanied by significant accumulation of succinyl-CoA, which in turn perturbed lysine succinylation in the proteome. Although cell wall structure and cross-linking were unchanged, significantly increased succinylation of the major autolysin Atl, which was the most succinylated protein in the proteome, was accompanied by near complete repression of autolytic activity. These findings link central metabolism and levels of succinyl-CoA to the regulation of β-lactam antibiotic resistance in MRSA through succinylome-mediated control of two interlinked cell wall phenotypes. Drug-mediated interference of the SucCD-controlled succinylome may help overcome β-lactam resistance

Full Activation of Enterococcus faecalis Gelatinase by a C-Terminal Proteolytic Cleavage▿ † ‡

Enterococci account for nearly 10% of all nosocomial infections and constitute a significant treatment challenge due to their multidrug resistance properties. One of the well-studied virulence factors of Enterococcus faecalis is a secreted bacterial protease, termed gelatinase, which has been shown to contribute to the process of biofilm formation. Gelatinase belongs to the M4 family of bacterial zinc metalloendopeptidases, typified by thermolysin. Gelatinase is synthesized as a preproenzyme consisting of a signal sequence, a putative propeptide, and then the mature enzyme. We determined that the molecular mass of the mature protein isolated from culture supernatant was 33,030 Da, which differed from the predicted molecular mass, 34,570 Da, by over 1,500 Da. Using N-terminal sequencing, we confirmed that the mature protein begins at the previously identified sequence VGSEV, thus suggesting that the 1,500-Da molecular mass difference resulted from a C-terminal processing event. By using mutants with site-directed mutations within a predicted C-terminal processing site and mutants with C-terminal deletions fused to a hexahistidine tag, we determined that the processing site is likely to be between residues D304 and I305 and that it requires the Q306 residue. The results suggest that the E. faecalis gelatinase requires C-terminal processing for full activation of protease activity, making it a unique enzyme among the members of the M4 family of proteases of gram-positive bacteria