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OBJECTIVE: Our previous studies have shown that murine dendritic cells (DCs) genetically modified to express interleukin-4 (IL-4) reduce the incidence and severity of murine collagen-induced arthritis. The present studies were performed to assess the immunoregulatory mechanisms underlying this response, by assessing the effects of IL-4 DCs on cytokine production by subsets of T helper cells. METHODS: Male DBA mice ages 6-8 weeks old were immunized with type II collagen. Splenic T cells obtained during the initiation phase and the end stage of arthritis were cultured with IL-4 DCs or untransduced DCs in the presence of collagen rechallenge. Interferon-gamma (IFNgamma) and IL-17 responses were measured. Antibodies to IL-4, IL-12, and IL-23, and recombinant IL-4, IL-12, and IL-23 were used to further study the regulation of T cell cytokine production by IL-4 DCs. RESULTS: Splenic T cells obtained during the initiation phase of arthritis produced less IL-17 when cultured in the presence of IL-4 DCs, despite their production of increased quantities of other proinflammatory cytokines (IFNgamma and tumor necrosis factor). T cell IL-17 production after collagen rechallenge was not inhibited by a lack of IL-23, since IL-4-mediated suppression of IL-17 was not reconstituted by IL-23, an otherwise potent inducer of IL-17 production by T cells. Although IL-4 DCs can produce increased quantities of IL-12 and IFNgamma, suppression of IL-17 production by IL-4 DCs was independent of both. While IL-17 production by T cells obtained during the initiation phase of arthritis was regulated by IL-4 DCs, IL-17 production by T cells obtained during end-stage arthritis was not altered. CONCLUSION: Our data suggest that IL-4 DCs exert a therapeutic effect on collagen-induced arthritis by targeting IL-17. IL-17 suppression by IL-4 DCs is robust and is not reversed by IL-23. Timing might be important in IL-17-targeted therapy, since IL-17 production by T cells obtained during end-stage arthritis did not respond to suppression by IL-4 DCs. [ABSTRACT FROM AUTHOR]

Fast and reliable identification of pathogenic bacteria is of upmost importance to human health and safety. Methods that are currently used in clinical practice are often time consuming, require expensive equipment, trained personnel, and therefore have limited applications in low resource environments. Molecular identification methods address some of these shortcomings. At the same time, they often use antibodies, their fragments, or other biomolecules as recognition units, which makes such tests specific to a particular target. In contrast, array-based methods use a combination of reporters that are not specific to a single pathogen. These methods provide a more data-rich and universal response that can be used for identification of a variety of bacteria of interest. In this report, we demonstrate the application of the excitation-emission spectroscopy of an environmentally sensitive fluorescent dye for identification of pathogenic bacterial species. 2-(4'-Dimethylamino)-3-hydroxyflavone (DMAF) interacts with the bacterial cell envelope resulting in a distinct spectral response that is unique to each bacterial species. The dynamics of dye-bacteria interaction were thoroughly investigated, and the limits of detection and identification were determined. Neural network classification algorithm was used for pattern recognition analysis and classification of spectral data. The sensor successfully discriminated between eight representative pathogenic bacteria, achieving a classification accuracy of 85.8% at the species level and 98.3% at the Gram status level. The proposed method based on excitation-emission spectroscopy of an environmentally sensitive fluorescent dye is a powerful and versatile diagnostic tool with high accuracy in identification of bacterial pathogens., Competing Interests: The authors declare no conflicts., (This journal is © The Royal Society of Chemistry.)

Genome sequencing has demonstrated that Staphylococcus aureus encodes arginine biosynthetic genes argDCJBFGH synthesizing proteins that mediate arginine biosynthesis using glutamate as a substrate. Paradoxically, however, S. aureus does not grow in a defined, glutamate-replete medium lacking arginine and glucose (CDM-R). Studies from our laboratory have found that specific mutations are selected by S. aureus that facilitate growth in CDM-R. However, these selected mutants synthesize arginine utilizing proline as a substrate rather than glutamate. In this study, we demonstrate that the ectopic expression of the argDCJB operon supports the growth of S. aureus in CDM-R, thus documenting the functionality of this pathway. Furthermore, suppressor mutants of S. aureus JE2 putA::Tn, which is defective in synthesizing arginine from proline, were selected on CDM-R agar. Genome sequencing revealed that these mutants had compensatory mutations within both spoVG , encoding an ortholog of the Bacillus subtilis stage V sporulation protein, and sarA, encoding the staphylococcal accessory regulator. Transcriptional studies document that argD expression is significantly increased when JE2 spoVG sarA was grown in CDM-R. Lastly, we found that a mutation in ahrC was required to induce argD expression in JE2 spoVG sarA when grown in an arginine-replete medium (CDM), suggesting that AhrC also functions to repress argDCJB in an arginine-dependent manner. In conclusion, these data indicate that the argDCJB operon is functional when transcribed in vitro and that SNPs within potential putative regulatory proteins are required to alleviate the repression.IMPORTANCEAlthough Staphylococcus aureus has the capability to synthesize all 20 amino acids, it is phenotypically auxotrophic for several amino acids including arginine. This work identifies putative regulatory proteins, including SpoVG, SarA, and AhrC, that function to inhibit the arginine biosynthetic pathways using glutamate as a substrate. Understanding the ultimate mechanisms of why S. aureus is selected to repress arginine biosynthetic pathways even in the absence of arginine will add to the growing body of work assessing the interactions between metabolism and S. aureus pathogenesis., Competing Interests: The authors declare no conflict of interest.

Under conditions of glucose excess, aerobically growing bacteria predominantly direct carbon flux towards acetate fermentation, a phenomenon known as overflow metabolism or the bacterial 'Crabtree effect'. Numerous studies of the major acetate-generating pathway, the Pta-AckA, revealed its important role in bacterial fitness through the control of central metabolism to sustain balanced growth and cellular homeostasis. In this work, we highlight the contribution of the Pta-AckA pathway to fitness of the spore-forming bacterium, Bacillus anthracis We demonstrate that disruption of the Pta-AckA pathway causes a drastic growth reduction in the mutants and alters the metabolic and energy status of the cells. Our results revealed that inactivation of the Pta-AckA pathway increases the glucose consumption rate, affects intracellular ATP, NAD + and NADH levels and leads to a metabolic block at the pyruvate and acetyl-CoA nodes. Consequently, accumulation of intracellular acetyl-CoA and pyruvate forces bacteria to direct carbon into the TCA and/or glyoxylate cycles as well as fatty acid and poly(3-hydroxybutyrate) (PHB) biosynthesis pathways. Notably, the presence of phosphate butyryltransferase in B. anthracis partially compensates for the loss of phosphotransacetylase activity. Furthermore, overexpression of the ptb gene not only eliminates the negative impact of the pta mutation on B. anthracis fitness, but also restores normal growth in the pta mutant of the non-butyrate-producing bacterium, Staphylococcus aureus Taken together, the results of this study demonstrate the importance of the Pta-AckA pathway for B. anthracis fitness by revealing its critical contribution to the maintenance of metabolic homeostasis during aerobic growth under conditions of carbon overflow. IMPORTANCE B. anthracis , the etiologic agent of anthrax, is a highly pathogenic, spore-forming bacterium that causes acute, life-threatening disease in both humans and livestock. A greater understanding of the metabolic determinants governing fitness of B. anthracis is essential for the development of successful therapeutic and vaccination strategies aimed at lessening the potential impact of this important biodefense pathogen. This study is the first to demonstrate the vital role of the Pta-AckA pathway in preserving energy and metabolic homeostasis in B. anthracis under conditions of carbon overflow, therefore, highlighting this pathway as a potential therapeutic target for drug discovery. Overall, the results of this study provide important insight into understanding the metabolic processes and requirements driving rapid B. anthracis proliferation during vegetative growth., (Copyright © 2021 American Society for Microbiology.)

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.

Oxygen consumption is a fundamental characteristic of staphylococcal physiology reflecting the energy and metabolic state of the bacterial cell. During aerobic growth, oxygen consumption rates (OCR) depend on nutrient availability and vary at different growth stages. The measurement of oxygen consumption rates provides a versatile tool to characterize the impact of various mutations, environmental cues, and antibiotics on bacterial growth and fitness. In this chapter, we describe a MitoXpress ® Xtra-based oxygen consumption assay for fast and reliable determination of respiration rates in Staphylococcus aureus. This highly reproducible and simple method requires a minimal set of reagents and allows rapid screening of multiple samples through real-time determination of the OCR with an oxygen-sensing probe and fluorescence plate reader., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

The intricate process of biofilm formation in the human pathogen Staphylococcus aureus involves distinct stages during which a complex mixture of matrix molecules is produced and modified throughout the developmental cycle. Early in biofilm development, a subpopulation of cells detaches from its substrate in an event termed "exodus" that is mediated by SaePQRS-dependent stochastic expression of a secreted staphylococcal nuclease, which degrades extracellular DNA within the matrix, causing the release of cells and subsequently allowing for the formation of metabolically heterogenous microcolonies. Since the SaePQRS regulatory system is involved in the transcriptional control of multiple S. aureus virulence factors, the expression of several additional virulence genes was examined within a developing biofilm by introducing fluorescent gene reporter plasmids into wild-type S. aureus and isogenic regulatory mutants and growing these strains in a microfluidic system that supplies the bacteria with a constant flow of media while simultaneously imaging developing biofilms in 5-min intervals. This study demonstrated that multiple virulence genes, including nuc , were expressed stochastically within a specialized subpopulation of cells in nascent biofilms. We demonstrated that virulence genes regulated by SaePQRS were stochastically expressed in nearly all strains examined whereas Agr-regulated genes were expressed more homogenously within maturing microcolonies. The commonly used Newman strain contains a variant of SaeS (SaeS P ) that confers constitutive kinase activity to the protein and caused this strain to lack the stochastic expression pattern observed in other strain backgrounds. Importantly, repair of the SaeS P allele resulting in reversion to the well-conserved SaeS L allele found in other strains restored stochastic expression in this strain. IMPORTANCE Staphylococcus aureus is an important human pathogen capable of colonizing diverse tissue types and inducing severe disease in both immunocompromised and otherwise healthy individuals. Biofilm infections caused by this bacterial species are of particular concern because of their persistence, even in the face of intensive therapeutic intervention. The results of the current study demonstrate the stochastic nature of Sae-mediated virulence gene expression in S. aureus and indicate that this regulatory system may function as a "bistable switch" in a manner similar to that seen with regulators controlling competence gene expression in Bacillus subtilis and persister cell formation in Escherichia coli The results of this study provide a new perspective on the complex mechanisms utilized by S. aureus during the establishment of infections., (Copyright © 2020 DelMain et al.)

The death and lysis of a subpopulation of Staphylococcus aureus cells during biofilm development benefit the whole bacterial population through the release of an important component of the biofilm matrix, extracellular DNA. Previously, we have demonstrated that these processes are affected by the gene products of the cidABC operon, the expression of which is controlled by the LysR-type transcriptional regulator, CidR. In this study, we characterized cis - and trans -acting elements essential for the induction of the cidABC operon. In addition to a CidR-binding site located within the cidABC promoter region, sequence analysis revealed the presence of a putative catabolite responsive element ( cre box), suggestive of the involvement of the catabolite control protein A (CcpA) in the regulation of cidABC expression. This was confirmed using electrophoretic mobility shift assays and real-time reverse transcriptase PCR analysis demonstrating the direct positive control of cidABC transcription by the master regulator of carbon metabolism. Furthermore, the importance of CcpA and the identified cre site for the induction of the cidABC operon was demonstrated by examining the expression of P cidABC -lacZ reporter fusions in various mutant strains in which the genes involved in carbon metabolism and carbon catabolite repression were disrupted. Together the results of this study demonstrate the necessity of both transcriptional regulators, CidR and CcpA, for the induction of the cidABC operon and reveal the complexity of molecular interactions controlling its expression. IMPORTANCE This work focuses on the characterization of cis - and trans -acting elements essential for the induction of the cidABC operon in S. aureus The results of this study are the first to demonstrate the synergistic control of cidABC expression by transcriptional regulators CidR and CcpA during carbohydrate metabolism. We established that the full induction of cidABC expression depends on the metabolic state of bacteria and requires both CidR and CcpA. Together, these findings delineate regulatory control of cidABC expression under different metabolic conditions and provide important new insights into our understanding of cell death mechanisms during biofilm development in S. aureus ., (Copyright © 2019 American Society for Microbiology.)

Transposon mutagenesis is one of the most widely used strategies to generate a large number of random mutations within a bacterial genome and then to precisely identify the mutated sites. The generation of sequence-defined transposon mutant libraries that are composed of a collection of different mutants, each containing a single transposon insertion mutation within nearly all of the nonessential genes within the genome, is a rapid and reliable way to enhance the study of gene function. In this chapter, we describe the process to generate a sequence-defined transposon mutant library in Staphylococcus aureus utilizing the mariner-based bursa aurealis transposon.

Numerous bacteria accumulate poly(3-hydroxybutyrate) (PHB) as an intracellular reservoir of carbon and energy in response to imbalanced nutritional conditions. In Bacillus spp., where PHB biosynthesis precedes the formation of the dormant cell type called the spore (sporulation), the direct link between PHB accumulation and efficiency of sporulation was observed in multiple studies. Although the idea of PHB as an intracellular carbon and energy source fueling sporulation was proposed several decades ago, the mechanisms underlying PHB contribution to sporulation have not been defined. Here, we demonstrate that PHB deficiency impairs Bacillus anthracis sporulation through diminishing the energy status of the cells and by reducing carbon flux into the tricarboxylic acid (TCA) cycle and de novo lipid biosynthesis. Consequently, this metabolic imbalance decreased biosynthesis of the critical components required for spore integrity and resistance, such as dipicolinic acid (DPA) and the spore's inner membrane. Supplementation of the PHB deficient mutant with exogenous fatty acids overcame these sporulation defects, highlighting the importance of the TCA cycle and lipid biosynthesis during sporulation. Combined, the results of this work reveal the molecular mechanisms of PHB contribution to B. anthracis sporulation and provide valuable insight into the metabolic requirements for this developmental process in Bacillus species., (© 2017 John Wiley & Sons Ltd.)

Community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) has become a severe health concern because of its treatment difficulties. Herein, we report the synthesis and biological evaluation of two phenazine natural products and a series of phenazines that show promising activities against MRSA with MIC values in the low micromolar range. Basic studies revealed that these compounds are bacteriostatic agents. The most active compound also displayed promising IC50 values against HaCat cells. Finally, a QSAR model was developed to understand the key structural features of the molecules., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Objectives: The increasing threat of drug-resistant bacteria establishes a continuing need for the development of new strategies to fight infection. We examine the inhibition of the essential single-stranded DNA-binding proteins (SSBs) SSBA and SSBB as a potential antimicrobial therapy due to their importance in DNA replication, activating the SOS response and promoting competence-based mechanisms of resistance by incorporating new DNA., Methods: Purified recombinant SSBs from Gram-positive (Staphylococcus aureus and Bacillus anthracis) and Gram-negative (Escherichia coli and Francisella tularensis) bacteria were assessed in a high-throughput screen for inhibition of duplex DNA unwinding by small molecule inhibitors. Secondary electrophoretic mobility shift assays further validated the top hits that were then tested for MICs using in vitro assays., Results: We have identified compounds that show cross-reactivity in vitro, as well as inhibition of both F. tularensis and B. anthracis SSBA. Five compounds were moderately toxic to at least two of the four bacterial strains in vivo, including two compounds that were selectively non-toxic to human cells, 9-hydroxyphenylfluoron and purpurogallin. Three of the SSBA inhibitors also inhibited S. aureus SSBB in Gram-positive bacteria., Conclusions: Results from our study support the potential for SSB inhibitors as broad-spectrum antibacterial agents, with dual targeting capabilities against Gram-positive bacteria., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Staphylococcus aureus (SA) is a major cause of hospital- and community-associated bacterial infections in the U.S. and around the world. These infections have become increasingly difficult to treat due to the propensity to develop antibiotic resistance and form biofilm. To date, no antibiofilm agents are available for clinical use. To add to the repertoire of antibiotics for clinical use and to provide novel agents for combating both SA and biofilm infections, we previously reported marinopyrroles as potent anti-SA agents. In this study, we used fragment-based and bioisostere approaches to design and synthesize a series of novel fluorinated pyrrolomycins for the first time, performed analyses of their physicochemical and drug-like properties, and investigated structure activity relationships and pharmacokinetics. These promising fluorinated pyrrolomycins demonstrate potent antibacterial activity against SA with favorable drug-like properties and pharmacokinetic profiles. Importantly, these compounds kill staphylococcal biofilm-associated cells with a lack of mammalian cell cytotoxicity and no occurrence of bacterial resistance. Our novel fluorinated pyrrolomycin 4 has a clogP value of 4.1, an MIC of 73 ng/mL, MBC of 4 μg/mL, kill staphylococcal-associated biofilm at 8 μg/mL, bioavailability of 35%, and the elimination half-life of 6.04 h and 6.75 h by intravenous and oral administration, respectively. This is the first report of comprehensive drug discovery studies on pyrrolomycin-based antibiotics., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Previous studies showed that sub-MIC levels of β-lactam antibiotics stimulate biofilm formation in most methicillin-resistant Staphylococcus aureus (MRSA) strains. Here, we investigated this process by measuring the effects of sub-MIC amoxicillin on biofilm formation by the epidemic community-associated MRSA strain USA300. We found that sub-MIC amoxicillin increased the ability of USA300 cells to attach to surfaces and form biofilms under both static and flow conditions. We also found that USA300 biofilms cultured in sub-MIC amoxicillin were thicker, contained more pillar and channel structures, and were less porous than biofilms cultured without antibiotic. Biofilm formation in sub-MIC amoxicillin correlated with the production of extracellular DNA (eDNA). However, eDNA released by amoxicillin-induced cell lysis alone was evidently not sufficient to stimulate biofilm. Sub-MIC levels of two other cell wall-active agents with different mechanisms of action-d-cycloserine and fosfomycin-also stimulated eDNA-dependent biofilm, suggesting that biofilm formation may be a mechanistic adaptation to cell wall stress. Screening a USA300 mariner transposon library for mutants deficient in biofilm formation in sub-MIC amoxicillin identified numerous known mediators of S. aureus β-lactam resistance and biofilm formation, as well as novel genes not previously associated with these phenotypes. Our results link cell wall stress and biofilm formation in MRSA and suggest that eDNA-dependent biofilm formation by strain USA300 in low-dose amoxicillin is an inducible phenotype that can be used to identify novel genes impacting MRSA β-lactam resistance and biofilm formation., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Transposon mutagenesis is a genetic process that involves the random insertion of transposons into a genome resulting in the disruption of function of the genes in which they insert. Identification of the insertion sites through DNA sequencing allows for the identification of the genes disrupted and the creation of "libraries" containing a collection of mutants in which a large number of the nonessential genes have been disrupted. These mutant libraries have been a great resource for investigators to understand the various biological functions of individual genes, including those involved in metabolism, antibiotic susceptibility, and pathogenesis. Here, we describe the detailed methodologies for constructing a sequence defined transposon mutant library in both Staphylococcus aureus and S. epidermidis using the mariner-based transposon, bursa aurealis.

Staphylococcus aureus nitrosative stress resistance is due in part to flavohemoprotein (Hmp). Although hmp is present in all sequenced S. aureus genomes, 37% of analyzed strains also contain nor, encoding a predicted quinol-type nitric oxide (NO) reductase (saNOR). DAF-FM staining of NO-challenged wild-type, nor, hmp and nor hmp mutant biofilms suggested that Hmp may have a greater contribution to intracellular NO detoxification relative to saNOR. However, saNOR still had a significant impact on intracellular NO levels and complemented NO detoxification in a nor hmp mutant. When grown as NO-challenged static (low-oxygen) cultures, hmp and nor hmp mutants both experienced a delay in growth initiation, whereas the nor mutant's ability to initiate growth was comparable with the wild-type strain. However, saNOR contributed to cell respiration in this assay once growth had resumed, as determined by membrane potential and respiratory activity assays. Expression of nor was upregulated during low-oxygen growth and dependent on SrrAB, a two-component system that regulates expression of respiration and nitrosative stress resistance genes. High-level nor promoter activity was also detectable in a cell subpopulation near the biofilm substratum. These results suggest that saNOR contributes to NO-dependent respiration during nitrosative stress, possibly conferring an advantage to nor+ strains in vivo., (© 2015 John Wiley & Sons Ltd.)

Recent studies have demonstrated that expression of the Staphylococcus aureus lrgAB operon is specifically localized within tower structures during biofilm development. To gain a better understanding of the mechanisms underlying this spatial control of lrgAB expression, we carried out a detailed analysis of the LytSR two-component system. Specifically, a conserved aspartic acid (Asp53) of the LytR response regulator was shown to be the target of phosphorylation, which resulted in enhanced binding to the lrgAB promoter and activation of transcription. In addition, we identified His390 of the LytS histidine kinase as the site of autophosphorylation and Asn394 as a critical amino acid involved in phosphatase activity. Interestingly, LytS-independent activation of LytR was observed during planktonic growth, with acetyl phosphate acting as a phosphodonor to LytR. In contrast, mutations disrupting the function of LytS prevented tower-specific lrgAB expression, providing insight into the physiologic environment within these structures. In addition, overactivation of LytR led to increased lrgAB promoter activity during planktonic and biofilm growth and a change in biofilm morphology. Overall, the results of this study are the first to define the LytSR signal transduction pathway, as well as determine the metabolic context within biofilm tower structures that triggers these signaling events., (© 2014 John Wiley & Sons Ltd.)

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.

Microbial virulence is a complex and often multifactorial phenotype, intricately linked to a pathogen's evolutionary trajectory. Toxicity, the ability to destroy host cell membranes, and adhesion, the ability to adhere to human tissues, are the major virulence factors of many bacterial pathogens, including Staphylococcus aureus. Here, we assayed the toxicity and adhesiveness of 90 MRSA (methicillin resistant S. aureus) isolates and found that while there was remarkably little variation in adhesion, toxicity varied by over an order of magnitude between isolates, suggesting different evolutionary selection pressures acting on these two traits. We performed a genome-wide association study (GWAS) and identified a large number of loci, as well as a putative network of epistatically interacting loci, that significantly associated with toxicity. Despite this apparent complexity in toxicity regulation, a predictive model based on a set of significant single nucleotide polymorphisms (SNPs) and insertion and deletions events (indels) showed a high degree of accuracy in predicting an isolate's toxicity solely from the genetic signature at these sites. Our results thus highlight the potential of using sequence data to determine clinically relevant parameters and have further implications for understanding the microbial virulence of this opportunistic pathogen.

Transposon mutant libraries are valuable resources to investigators studying bacterial species, including Staphylococcus epidermidis, which are difficult to genetically manipulate. Although sequence-defined transposon mutant libraries have been constructed in Staphylococcus aureus, no such library exists for S. epidermidis. Nevertheless, the study of Tn917-mediated mutations has been paramount in discovering unique aspects of S. epidermidis biology including initial adherence and accumulation during biofilm formation. Herein, we describe modifications to the methodology first described by Bae et al. to utilize the mariner-based transposon bursa aurealis to generate mutants in S. epidermidis strain 1457.

The Staphylococcus aureus cid and lrg operons play significant roles in the control of autolysis and accumulation of extracellular genomic DNA (eDNA) during biofilm development. Although the molecular mechanisms mediating this control are only beginning to be revealed, it is clear that cell death must be limited to a subfraction of the biofilm population. In the present study, we tested the hypothesis that cid and lrg expression varies during biofilm development as a function of changes in the availability of oxygen. To examine cid and lrg promoter activity during biofilm development, fluorescent reporter fusion strains were constructed and grown in a BioFlux microfluidic system, generating time-lapse epifluorescence images of biofilm formation, which allows the spatial and temporal localization of gene expression. Consistent with cid induction under hypoxic conditions, the cid::gfp fusion strain expressed green fluorescent protein predominantly within the interior of the tower structures, similar to the pattern of expression observed with a strain carrying a gfp fusion to the hypoxia-induced promoter controlling the expression of the lactose dehydrogenase gene. The lrg promoter was also expressed within towers but appeared more diffuse throughout the tower structures, indicating that it was oxygen independent. Unexpectedly, the results also demonstrated the existence of tower structures with different expression phenotypes and physical characteristics, suggesting that these towers exhibit different metabolic activities. Overall, the findings presented here support a model in which oxygen is important in the spatial and temporal control of cid expression within a biofilm and that tower structures formed during biofilm development exhibit metabolically distinct niches.

Unlabelled: To enhance the research capabilities of investigators interested in Staphylococcus aureus, the Nebraska Center for Staphylococcal Research (CSR) has generated a sequence-defined transposon mutant library consisting of 1,952 strains, each containing a single mutation within a nonessential gene of the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) isolate USA300. To demonstrate the utility of this library for large-scale screening of phenotypic alterations, we spotted the library on indicator plates to assess hemolytic potential, protease production, pigmentation, and mannitol utilization. As expected, we identified many genes known to function in these processes, thus validating the utility of this approach. Importantly, we also identified genes not previously associated with these phenotypes. In total, 71 mutants displayed differential hemolysis activities, the majority of which were not previously known to influence hemolysin production. Furthermore, 62 mutants were defective in protease activity, with only 14 previously demonstrated to be involved in the production of extracellular proteases. In addition, 38 mutations affected pigment formation, while only 7 influenced mannitol fermentation, underscoring the sensitivity of this approach to identify rare phenotypes. Finally, 579 open reading frames were not interrupted by a transposon, thus providing potentially new essential gene targets for subsequent antibacterial discovery. Overall, the Nebraska Transposon Mutant Library represents a valuable new resource for the research community that should greatly enhance investigations of this important human pathogen., Importance: Infections caused by Staphylococcus aureus cause significant morbidity and mortality in both community and hospital environments. Specific-allelic-replacement mutants are required to study the biology of this organism; however, this process is costly and time-consuming. We describe the construction and validation of a sequence-defined transposon mutant library available for use by the scientific community through the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) strain repository. In addition, complementary resources, including a website (http://app1.unmc.edu/fgx/) and genetic tools that expedite the allelic replacement of the transposon in the mutants with useful selectable markers and fluorescent reporter fusions, have been generated. Overall, this library and associated tools will have a significant impact on studies investigating S. aureus pathogenesis and biology and serve as a useful paradigm for the study of other bacterial systems.

The Staphylococcus aureus cid and lrg operons are known to be involved in biofilm formation by controlling cell lysis and the release of genomic DNA, which ultimately becomes a structural component of the biofilm matrix. Although the molecular mechanisms controlling cell death and lysis are unknown, it has been hypothesized that the cidA and lrgA genes encode holin- and antiholin-like proteins and function to regulate these processes similarly to bacteriophage-induced death and lysis. In this study, we focused on the biochemical and molecular characterization of CidA and LrgA with the goal of testing the holin model. First, membrane fractionation and fluorescent protein fusion studies revealed that CidA and LrgA are membrane-associated proteins. Furthermore, similarly to holins, CidA and LrgA were found to oligomerize into high-molecular-mass complexes whose formation was dependent on disulfide bonds formed between cysteine residues. To determine the function of disulfide bond-dependent oligomerization of CidA, an S. aureus mutant in which the wild-type copy of the cidA gene was replaced with the cysteine mutant allele was generated. As determined by β-galactosidase release assays, this mutant exhibited increased cell lysis during stationary phase, suggesting that oligomerization has a negative impact on this process. When analyzed for biofilm development and maturation, this mutant displayed increased biofilm adhesion in a static assay and a greater amount of dead-cell accumulation during biofilm maturation. These studies support the model that CidA and LrgA proteins are bacterial holin-/antiholin-like proteins that function to control cell death and lysis during biofilm development.

Introduction: Rheumatoid arthritis (RA) is now suspected to be driven by pathogenic Th17 cells that secrete interleukin (IL)-17 and can be regulated by IL-4. A single-nucleotide polymorphism (SNP), I50V, in the coding region of the human IL-4 receptor (IL-4R) is associated with rapid development of erosive disease in RA. The present study was undertaken to determine whether this SNP renders the IL-4R less able to transduce signals that regulate IL-17 production., Methods: Peripheral blood mononuclear cells were activated under Th17-stimulating conditions in the presence or absence of IL-4, and IL-17 production was measured by both enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Serum IL-17 was also measured by ELISA. Paired comparisons were performed using the two-tailed t-test. IL-4 receptor gene alleles were determined by polymerase chain reaction., Results: In healthy individuals, IL-4 significantly inhibited IL-17 production by cells from subjects with the I/I genotype (P = 0.0079) and the I/V genotype (P = 0.013), but not the V/V genotype (P > 0.05). In a cross-sectional sample of patients with established RA, the magnitude of the in vitro effect of IL-4 was lower and was not associated with a specific IL-4R allele. Serum IL-17 levels were higher in RA patients than in healthy individuals, as was the percentage of CD4+ cells that produced IL-17., Conclusions: These results indicate that an inherited polymorphism of the IL-4R controls the ability of the human immune system to regulate the magnitude of IL-17 production. However, in established RA, this pattern may be altered, possibly due to secondary effects of both RA itself as well as immunomodulatory medications. Ineffective control of Th17 immune responses is a potential mechanism to explain why IL-4R is an important severity gene in RA, but this issue will require careful study of a cohort of new-onset RA patients.

The par locus of the Enterococcus faecalis plasmid pAD1 is an RNA-regulated addiction module encoding the peptide toxin Fst. Homology searches revealed that Fst belongs to a family of at least nine related peptides encoded on the chromosomes and plasmids of six different Gram-positive bacterial species. Comparison of an alignment of these peptides with the results of a saturation mutagenesis analysis indicated regions of the peptides important for biological function. Examination of the genetic context of the fst genes revealed that all of these peptides are encoded within par-like loci with conserved features similar to pAD1 par. All four Ent. faecalis family members were demonstrated to produce the expected toxin-encoding and regulatory RNA products. The locus from the Ent. faecalis plasmid pAMS1 was demonstrated to function as an addiction module and Fst was shown to be toxic to Staphylococcus aureus, suggesting that a plasmid-encoded module in that species is performing the same function. Thus, the pAD1-encoded par locus appears to be the prototype of a family of related loci found in several Gram-positive species.

Recent studies have demonstrated a role for Staphylococcus aureus cidA-mediated cell lysis and genomic DNA release in biofilm adherence. The current study extends these findings by examining both temporal and additional genetic factors involved in the control of genomic DNA release and degradation during biofilm maturation. Cell lysis and DNA release were found to be critical for biofilm attachment during the initial stages of development and the released DNA (eDNA) remained an important matrix component during biofilm maturation. This study also revealed that an lrgAB mutant exhibits increased biofilm adherence and matrix-associated eDNA consistent with its proposed role as an inhibitor of cidA-mediated lysis. In flow-cell assays, both cid and lrg mutations had dramatic effects on biofilm maturation and tower formation. Finally, staphylococcal thermonuclease was shown to be involved in biofilm development as a nuc mutant formed a thicker biofilm containing increased levels of matrix-associated eDNA. Together, these findings suggest a model in which the opposing activities of the cid and lrg gene products control cell lysis and genomic DNA release during biofilm development, while staphylococcal thermonuclease functions to degrade the eDNA, possibly as a means to promote biofilm dispersal.

Fibroblast-like synoviocytes (FLS) and T cells can activate each other in vitro, and in vivo interactions between these cells may be important in rheumatoid arthritis (RA), yet FLS lack significant expression of CD28 ligands. We sought to identify molecules homologous to CD28 ligands that are strongly expressed by FLS, and documented strong B7-H3 expression on FLS and by fibroblasts of other tissues, which was unaffected by a variety of cytokines. Western blot analysis of FLS lysates showed predominant expression of the larger, four Ig-like domain isoform of B7-H3. Immunohistological sections of RA synovial tissue showed strong staining for B7-H3 on FLS. Cells expressing B7-H3 were distinct from but in close proximity to cells that expressed CD45, CD20, and CD3. Confocal microscopy of FLS and T cell cocultures showed localization of B7-H3 in the region of the T cell-FLS contact point, but distinct from the localization of T cell CD11a/CD18 (LFA-1) and FLS CD54 (ICAM-1). Reduction of B7-H3 expression on FLS by RNA interference affected interactions of FLS with resting T cells or cytokine-activated T cells. Resting T cells showed increased production of TNF-alpha, IFN-gamma, and IL-2, whereas cytokine-activated T cells showed reduced cytokine production relative to control. However, cytokine production by T cells activated through their TCR was not notably altered by knock down of B7-H3. These observations suggest that B7-H3 may be important for the interactions between FLS and T cells in RA, as well as other diseases, and the outcome of such interactions depends on the activation state of the T cell.

The mechanism of fibroblast-like synoviocyte (FLS) transformation into an inflammatory phenotype in rheumatoid arthritis (RA) is not fully understood. FLS interactions with invading leukocytes, particularly T cells, are thought to be a critical component of this pathological process. Resting T cells and T cells activated through the T-cell receptor have previously been shown to induce inflammatory cytokine production by FLS. More recently, a distinct population of T cells has been identified in RA synovium that phenotypically resembles cytokine-activated T (Tck) cells. Using time lapse microscopy, the interactions of resting, superantigen-activated, and cytokine-activated T cells with FLS were visualized. Rapid and robust adhesion of Tck and superantigen-activated T cells to FLS was observed that resulted in flattening of the T cells and a crawling movement on the FLS surface. Tck also readily activated FLS to produce interleukin IL-6 and IL-8 in a cell contact-dependent manner that was enhanced by exogenous IL-17. Although LFA-1 and ICAM-1 co-localized at the Tck-FLS synapse, blocking the LFA-1/ICAM-1 interaction did not substantially inhibit Tck effector function. However, antibody blocking of membrane tumor necrosis factor (TNF)-alpha on the Tck surface did inhibit FLS cytokine production, thus illustrating a novel mechanism for involvement of TNF-alpha in cell-cell interactions in RA synovium and for the effectiveness of TNF-alpha blockade in the treatment of RA.

The Staphylococcus aureus cidA and lrgA genes have been shown to affect cell lysis under a variety of conditions during planktonic growth. It is hypothesized that these genes encode holins and antiholins, respectively, and may serve as molecular control elements of bacterial cell lysis. To examine the biological role of cell death and lysis, we studied the impact of the cidA mutation on biofilm development. Interestingly, this mutation had a dramatic impact on biofilm morphology and adherence. The cidA mutant (KB1050) biofilm exhibited a rougher appearance compared with the parental strain (UAMS-1) and was less adherent. Propidium iodide staining revealed that KB1050 accumulated more dead cells within the biofilm population relative to UAMS-1, indicative of reduced cell lysis. In agreement with this finding, quantitative real-time PCR experiments demonstrated the presence of 5-fold less genomic DNA in the KB1050 biofilm relative to UAMS-1. Furthermore, treatment of the UAMS-1 biofilm with DNase I caused extensive cell detachment, whereas similar treatment of the KB1050 biofilm had only a modest effect. These results demonstrate that cidA-controlled cell lysis plays a significant role during biofilm development and that released genomic DNA is an important structural component of S. aureus biofilm.

Objective: To assess the ability of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) to function as antigen-presenting cells (APCs) for arthritogenic autoantigens found within inflamed joint tissues., Methods: Human class II major histocompatibility complex (MHC)-typed FLS were used as APCs for murine class II MHC-restricted CD4 T cell hybridomas. Interferon-gamma (IFNgamma)-treated, antigen-loaded FLS were cocultured with T cell hybridomas specific for immunodominant portions of human cartilage gp-39 (HC gp-39) or human type II collagen (CII). T cell hybridoma activation was measured by enzyme-linked immunosorbent assay of culture supernatants for interleukin-2. Both synthetic peptide and synovial fluid (SF) were used as sources of antigen. APC function in cocultures was inhibited by using blocking antibodies to human class II MHC, CD54, or CD58, or to murine CD4, CD11a, or CD2., Results: Human FLS could present peptides from the autoantigens HC gp-39 and human CII to antigen-specific MHC-restricted T cell hybridomas. This response required pretreatment of FLS with IFNgamma, showed MHC restriction, and was dependent on human class II MHC and murine CD4 for effective antigen presentation. Furthermore, FLS were able to extract and present antigens found within human SF to both the HC gp-39 and human CII T cell hybridomas in an IFNgamma-dependent and MHC-restricted manner., Conclusion: RA FLS can function as APCs and are able to present peptides derived from autoantigens found within joint tissues to activated T cells in vitro. In the context of inflamed synovial tissues, FLS may be an important and hitherto overlooked subset of APCs that could contribute to autoreactive immune responses.

Objective: CD6, a cell surface glycoprotein expressed primarily on T cells, may function as a costimulatory molecule and may play a role in autoreactive immune responses. Recently, a CD6 ligand termed CD166 (previously known as activated leukocyte cell adhesion molecule [ALCAM]) has been identified and shown to be expressed on activated T cells, B cells, thymic epithelium, keratinocytes, and in rheumatoid arthritis synovial tissue. However, the results of functional studies have suggested the existence of a second CD6 ligand. The present study was undertaken to seek evidence for a second CD6 ligand on cultured synovial fibroblasts., Methods: Flow cytometric and biochemical techniques were applied, using anti-CD166 monoclonal antibody (mAb) and a recombinant CD6 fusion protein, to determine whether cultured synovial fibroblasts and other cell types expressed a non-ALCAM CD6 ligand., Results: CD14- fibroblastic synoviocytes showed greater binding of a recombinant CD6 fusion protein than of anti-ALCAM mAb. With interferon-gamma treatment of synovial fibroblasts, binding of both reagents increased, but this was more marked for binding of CD6 fusion protein. Exposure of synovial fibroblasts to other cytokines or to the superantigen staphylococcal enterotoxin A also regulated binding of CD6 fusion protein and anti-ALCAM mAb in a discordant manner. Immunoprecipitation of proteins from membrane extracts of synovial fibroblasts with a CD6-Ig fusion protein revealed a novel 130-kd band distinct from CD166; an identical molecule was also precipitated from membranes of HBL-100 tumor cells., Conclusion: Taken together with previous data regarding CD6 and CD166 function, the present findings strongly suggest the existence of a second CD6 ligand distinct from CD166, which can be expressed by synovial fibroblasts as well as other cells.

The cooperative effects of the GPIa 807TT, MTHFR 677TT and prothrombin 20210GA genotypes with the FV Leiden 1691GA (FVL) genotype were evaluated by comparing these genotype frequencies in 77 asymptomatic and 156 symptomatic heterozygous FVL carriers. The GPIa 807TT and MTHFR 677TT genotypes did not segregate within the symptomatic FVL carrier group and did not contribute to venous thrombotic risk in this patient cohort. There was no difference in the prothrombin 20210GA genotype frequency between asymptomatic FVL carriers and a random Caucasian control group; however, the prothrombin 20210GA genotype was nearly 5 times as prevalent (19/156 v 2/77; P < 0.02) in the symptomatic FVL carriers (odds ratio 5.21; 95% confidence interval 1.20-47.62), demonstrating that this important prothrombotic risk factor acts synergistically with FVL.

Background: Sirolimus (formerly rapamycin) is an immunosuppressive agent that interferes with T-cell activation. After 2 individuals with psoriasis developed a capillary leak syndrome following treatment with oral sirolimus lesional skin cells and activated peripheral blood cells were analyzed for induction of apoptosis., Observations: A keratome skin specimen from 1 patient with sirolimus-induced capillary leak syndrome had a 2.3-fold increase in percentage of apoptotic cells (to 48%) compared with an unaffected sirolimus-treated patient with psoriasis (21%). Activated peripheral blood T cells from patients with psoriasis tended to exhibit greater spontaneous or dexamethasone-induced apoptosis than did normal T cells, particularly in the presence of sirolimus., Conclusions: Severe adverse effects of sirolimus include fever, anemia, and capillary leak syndrome. These symptoms may be the result of drug-induced apoptosis of lesional leukocytes, especially activated T lymphocytes, and possibly release of inflammatory mediators. Because patients with severe psoriasis may develop capillary leak from various systemic therapies, clinical monitoring is advisable for patients with inflammatory diseases who are treated with immune modulators.

Groups of Swiss ICR mice were fed 1000 ppm polybrominated biphenyls (FireMaster BP-6) in rodent chow for 4, 8, 11, and 14 days. Control groups were fed standard rodent chow without FireMaster BP-6. Animals were killed at the end of each feeding period and the livers examined by electron microscopy. EM changes noted were progressive increase in size of hepatocytes, a decrease in rough endoplasmic reticulum, a marked increase in smooth endoplasmic reticulum, mitochondrial degeneration, increased lysosomes, and a decrease in glycogen. In addition, there was increasing proliferation of microvilli in bile canaliculi with increasing feeding times. A group of mice fed 1000 ppm FireMaster BP-6 in rodent chow for 11 days had livers with a mean of 13.93% of total body weight as compared with 6.49% for the control group (P=0.02). Tissue distribution following ingestion of 100 ppm FireMaster BP-6 for 14 days was studied. Twelve weeks post-feeding, the tissue concentrations of hexabromobiphenyl in order of highest concentration to lowest were as follows: perithymic fat, perirenal fat, adrenal glands, thymus gland, liver and stomach.