Enterococcus faecalis capsular polysaccharide serotypes C and D and their contributions to host innate immune evasion

It has become increasingly difficult to treat infections caused by Enterococcus faecalis due to the high levels of intrinsic and acquired antibiotic resistances. However, few studies have explored the mechanisms that E. faecalis employs to circumvent the host innate immune response and establish infection. Capsule polysaccharides are important virulence factors that are associated with innate immune evasion. We demonstrate that capsule producing E. faecalis strains of either serotype C or D are more resistant to complement-mediated opsonophagocytosis compared to un-encapsulated strains using cultured macrophages (RAW 264.7). We show that differences in opsonophagocytosis are not due to variation in C3 deposition, but due to the ability of capsule to mask bound C3 from detection on the surface of E. faecalis. Similarly, E. faecalis capsule masks detection of lipoteichoic acid which correlates with decreased TNF-α production by cultured macrophages in the presence of encapsulated strains compared to unencapsulated strains. Our studies confirm the important role of the capsule as a virulence factor of E. faecalis, and provide several mechanisms by which the presence of the capsule influences evasion of the innate immune response, and suggest that the capsule could be a potential target for developing alternative therapies to treat E. faecalis infections

The ClpXP protease is dispensable for degradation of unfolded proteins in <i>Staphylococcus aureus</i>

Abstract In living cells intracellular proteolysis is crucial for protein homeostasis, and ClpP proteases are conserved between eubacteria and the organelles of eukaryotic cells. In Staphylococcus aureus, ClpP associates to the substrate specificity factors, ClpX and ClpC forming two ClpP proteases, ClpXP and ClpCP. To address how individual ClpP proteases impact cell physiology, we constructed a S. aureus mutant expressing ClpX with an I265E substitution in the ClpP recognition tripeptide of ClpX. This mutant cannot degrade established ClpXP substrates confirming that the introduced amino acid substitution abolishes ClpXP activity. Phenotypic characterization of this mutant showed that ClpXP activity controls cell size and is required for growth at low temperature. Cells expressing the ClpXI265E variant, in contrast to cells lacking ClpP, are not sensitive to heat-stress and do not accumulate protein aggregates showing that ClpXP is dispensable for degradation of unfolded proteins in S. aureus. Consistent with this finding, transcriptomic profiling revealed strong induction of genes responding to protein folding stress in cells devoid of ClpP, but not in cells lacking only ClpXP. In the latter cells, highly upregulated loci include the urease operon, the pyrimidine biosynthesis operon, the betA-betB operon, and the pathogenicity island, SaPI5, while virulence genes were dramatically down-regulated

An Integrated Computational and Experimental Study to Investigate Staphylococcus Aureus 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

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

Urease is an Essential Component of the Acid Response Network of Staphylococcus 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

Catabolic Ornithine Carbamoyltransferase Activity Facilitates Growth of Staphylococcus aureus in Defined Medium Lacking Glucose and Arginine

Previous studies have found that arginine biosynthesis in Staphylococcus aureus is repressed via carbon catabolite repression (CcpA), and proline is used as a precursor. Unexpectedly, however, robust growth of S. aureus is not observed in complete defined medium lacking both glucose and arginine (CDM-R). Mutants able to grow on agar-containing defined medium lacking arginine (CDM-R) were selected and found to contain mutations within ahrC, encoding the canonical arginine biosynthesis pathway repressor (AhrC), or single nucleotide polymorphisms (SNPs) upstream of the native arginine deiminase (ADI) operon arcA1B1D1C1. Reverse transcription-PCR (RT-PCR) studies found that mutations within ccpA or ahrC or SNPs identified upstream of arcA1B1D1C1 increased the transcription of both arcB1 and argGH, encoding ornithine carbamoyltransferase and argininosuccinate synthase/lyase, respectively, facilitating arginine biosynthesis. Furthermore, mutations within the AhrC homologue argR2 facilitated robust growth within CDM-R. Complementation with arcB1 or arcA1B1D1C1, but not argGH, rescued growth in CDM-R. Finally, supplementation of CDM-R with ornithine stimulated growth, as did mutations in genes (proC and rocA) that presumably increased the pyrroline-5-carboxylate and ornithine pools. Collectively, these data suggest that the transcriptional regulation of ornithine carbamoyltransferase and, in addition, the availability of intracellular ornithine pools regulate arginine biosynthesis in S. aureus in the absence of glucose. Surprisingly, ~50% of clinical S. aureus isolates were able to grow in CDM-R. These data suggest that S. aureus is selected to repress arginine biosynthesis in environments with or without glucose; however, mutants may be readily selected that facilitate arginine biosynthesis and growth in specific environments lacking arginine. IMPORTANCE Staphylococcus aureus can cause infection in virtually any niche of the human host, suggesting that it has significant metabolic versatility. Indeed, bioinformatic analysis suggests that it has the biosynthetic capability to synthesize all 20 amino acids. Paradoxically, however, it is conditionally auxotrophic for several amino acids, including arginine. Studies in our laboratory are designed to assess the biological function of amino acid auxotrophy in this significant pathogen. This study reveals that the metabolic block repressing arginine biosynthesis in media lacking glucose is the transcriptional repression of ornithine carbamoyltransferase encoded by arcB1 within the native arginine deiminase operon in addition to limited intracellular pools of ornithine. Surprisingly, approximately 50% of S. aureus clinical isolates can grow in media lacking arginine, suggesting that mutations are selected in S. aureus that allow growth in particular niches of the human host

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

The molecular control and biological implications of autolysis in enterococcus faecalis biofilm development

Doctor of PhilosophyDepartment of BiologyLynn E. HancockThe enterococci are gaining much notoriety as common nosocomial pathogens. One aspect of their pathogenesis, especially characteristic to infectious endocarditis and urinary tract infections, involves their ability to transition from the sessile state of existence to surface adherent structured communities called biofilms. Existence as biofilms, affords enterococci protection against a number of growth limiting challenges including antibiotic therapy and host immunity. In the current study a mechanistic role for two Fsr quorum-regulated extracellular proteases- gelatinase (GelE) and its cotranscribed serine protease (SprE), were explored in biofilm development of E. faecalis V583. Confocal imaging of biofilms suggested that GelE[superscript]– mutants were significantly reduced in biofilm biomass compared to V583, whereas the absence of SprE appeared to accelerate the progression of biofilm development. Culture supernatant and biofilm analysis confirmed that decreased biofilms observed in GelE[superscript]– mutants resulted from their inability to undergo autolysis and release extracellular DNA (eDNA) in planktonic and biofilm cultures, whereas SprE[superscript]– mutants produced significantly more eDNA as components of the biofilm matrix. The governing principle behind GelE mediated autolysis and eDNA release in E. faecalis V583 was demonstrated to be fratricide. GFP reporter assays of V583 populations confirmed that GBAP (gelatinase biosynthesis-activating pheromone encoded by fsrD) quorum non-responders (GelE[superscript]–SprE[superscript]–) were a minority subpopulation of prey cells susceptible to the targeted fratricidal action of the quorum responsive predatorial majority (GelE[superscript]+SprE[superscript]+). The killing action is dependent on GelE, and the GelE producer population is protected from self-destruction by the co-production of SprE as an immunity protein. Targeted gene inactivation and protein interaction studies demonstrate that extracellular proteases execute their characteristic effects following downstream interactions with the primary autolysin, AtlA. Finally, comparison of virulence effects of isogenic extracellular protease mutants (∆gelE, ∆sprE and ∆gelEsprE) relative to parental strain (V583) in a rabbit model of enterococcal endocarditis confirmed a critical role for GelE in the infection process. In conclusion, the data presented in this thesis are consistent with significant roles for GelE and SprE in biofilm mediated pathogenesis of enterococcal infections

Capsular Polysaccharide Production in Enterococcus faecalis and Contribution of CpsF to Capsule Serospecificity ▿ †

Many bacterial species produce capsular polysaccharides that contribute to pathogenesis through evasion of the host innate immune system. The gram-positive pathogen Enterococcus faecalis was previously reported to produce one of four capsule serotypes (A, B, C, or D). Previous studies describing the four capsule serotypes of E. faecalis were based on immunodetection methods; however, the underlying genetics of capsule production did not fully support these findings. Previously, it was shown that capsule production for serotype C (Maekawa type 2) was dependent on the presence of nine open reading frames (cpsC to cpsK). Using a novel genetic system, we demonstrated that seven of the nine genes in the cps operon are essential for capsule production, indicating that serotypes A and B do not make a capsular polysaccharide. In support of this observation, we showed that serotype C and D capsule polysaccharides mask lipoteichoic acid from detection by agglutinating antibodies. Furthermore, we determined that the genetic basis for the difference in antigenicity between serotypes C and D is the presence of cpsF in serotype C strains. High-pH anion-exchange chromatography with pulsed amperometric detection analysis of serotype C and D capsules indicated that cpsF is responsible for glucosylation of serotype C capsular polysaccharide in E. faecalis

Regulation of Autolysis-Dependent Extracellular DNA Release by Enterococcus faecalis Extracellular Proteases Influences Biofilm Development▿

Enterococci are major contributors of hospital-acquired infections and have emerged as important reservoirs for the dissemination of antibiotic resistance traits. The ability to form biofilms on medical devices is an important aspect of pathogenesis in the hospital environment. The Enterococcus faecalis Fsr quorum system has been shown to regulate biofilm formation through the production of gelatinase, but the mechanism has been hitherto unknown. Here we show that both gelatinase (GelE) and serine protease (SprE) contribute to biofilm formation by E. faecalis and provide clues to how the activity of these proteases governs this developmental process. Confocal imaging of biofilms suggested that GelE− mutants were significantly reduced in biofilm biomass compared to the parental strain, whereas the absence of SprE appeared to accelerate the progression of biofilm development. The phenotype observed in a SprE− mutant was linked to an observed increase in autolytic rate compared to the parental strain. Culture supernatant analysis and confocal microscopy confirmed the inability of mutants deficient in GelE to release extracellular DNA (eDNA) in planktonic and biofilm cultures, whereas cells deficient in SprE produced significantly more eDNA as a component of the biofilm matrix. DNase I treatment of E. faecalis biofilms reduced the accumulation of biofilm, implying a critical role for eDNA in biofilm development. In conclusion, our data suggest that the interplay of two secreted and coregulated proteases—GelE and SprE—is responsible for regulating autolysis and the release of high-molecular-weight eDNA, a critical component for the development of E. faecalis biofilms