Stimulation Strength Determined by Superantigen Dose Controls Subcellular Localization of FOXP3 Isoforms and Suppressive Function of CD4+CD25+FOXP3+ T Cells.

Staphylococcal superantigens induce massive activation of T cells and inflammation, leading to toxic shock syndrome. Paradoxically, increasing evidence indicates that superantigens can also induce immunosuppression by promoting regulatory T cell (Treg) development. In this study, we demonstrate that stimulation strength plays a critical role in superantigen-mediated induction of immunosuppressive human CD4+CD25+FOXP3+ T cells. Suboptimal stimulation by a low dose (1 ng/ml) of staphylococcal enterotoxin C1 (SEC1) led to de novo generation of Treg-like CD4+CD25+FOXP3+ T cells with strong suppressive activity. In contrast, CD4+CD25+ T cells induced by optimal stimulation with high-dose SEC1 (1 µg/ml) were not immunosuppressive, despite high FOXP3 expression. Signal transduction pathway analysis revealed differential activation of the PI3K signaling pathway and expression of PTEN in optimal and suboptimal stimulation with SEC1. Additionally, we identified that FOXP3 isoforms in Treg-like cells from the suboptimal condition were located in the nucleus, whereas FOXP3 in nonsuppressive cells from the optimal condition localized in cytoplasm. Sequencing analysis of FOXP3 isoform transcripts identified five isoforms, including a FOXP3 isoform lacking partial exon 3. Overexpression of FOXP3 isoforms confirmed that both an exon 2-lacking isoform and a partial exon 3-lacking isoform confer suppressive activity. Furthermore, blockade of PI3K in optimal stimulation conditions led to induction of suppressive Treg-like cells with nuclear translocation of FOXP3, suggesting that PI3K signaling impairs induction of Tregs in a SEC1 dose-dependent manner. Taken together, these data demonstrate that the strength of activation signals determined by superantigen dose regulates subcellular localization of FOXP3 isoforms, which confers suppressive functionality.

GLIS1 in Cancer-Associated Fibroblasts Regulates the Migration and Invasion of Ovarian Cancer Cells.

A cancer-associated fibroblasts (CAFs) are the most important players that modulate tumor aggressiveness. In this study, we aimed to identify CAF-related genes in ovarian serous carcinomas (OSC) that account for the high incidence and mortality of ovarian cancers (OCs) and to develop therapeutic targets for tumor microenvironment modulation. Here, we performed a microarray analysis of CAFs isolated from three metastatic and three nonmetastatic OSC tissues and compared their gene expression profiles. Among the genes increased in metastatic CAFs (mCAFs), GLIS1 (Glis Family Zinc Finger 1) showed a significant increase in both the gene mRNA and protein expression levels. Knockdown of GLIS1 in mCAFs significantly inhibited migration, invasion, and wound healing ability of OC cells. In addition, an in vivo study demonstrated that knockdown of GLIS1 in CAFs reduced peritoneal metastasis. Taken together, these results suggest that CAFs support migration and metastasis of OC cells by GLIS1 overexpression. It also indicates GLIS1 in CAFs might be a potential therapeutic target to inhibit OC metastasis.

Induction of Immunosuppressive CD8+CD25+FOXP3+ Regulatory T Cells by Suboptimal Stimulation with Staphylococcal Enterotoxin C1.

Superantigens (SAgs) produced by Staphylococcus aureus at high concentrations induce proliferation of T cells bearing specific TCR Vß sequences and massive cytokinemia that cause toxic shock syndrome. However, the biological relevance of SAgs produced at very low concentrations during asymptomatic colonization or chronic infections is not understood. In this study, we demonstrate that suboptimal stimulation of human PBMCs with a low concentration (1 ng/ml) of staphylococcal enterotoxin C1, at which half-maximal T cell proliferation was observed, induced CD8+CD25+ T cells expressing markers related to regulatory T cells (Tregs), such as IFN-γ, IL-10, TGF-ß, FOXP3, CD28, CTLA4, TNFR2, CD45RO, and HLA-DR. Importantly, these CD8+CD25+ T cells suppressed responder cell proliferation mediated in contact-dependent and soluble factor-dependent manners, involving galectin-1 and granzymes, respectively. In contrast, optimal stimulation of human PBMCs with a high concentration (1 µg/ml) of staphylococcal enterotoxin C1, at which maximal T cell proliferation was observed, also induced similar expression of markers related to Tregs, including FOXP3 in CD8+CD25+ cells, but these T cells were not functionally immunosuppressive. We further demonstrated that SAg-induced TCR Vß-restricted and MHC class II-restricted expansion of immunosuppressive CD8+CD25+ T cells is independent of CD4+ T cells. Our results suggest that the concentration of SAg strongly affects the functional characteristics of activated T cells, and low concentrations of SAg produced during asymptomatic colonization or chronic S. aureus infection induce immunosuppressive CD8+ Tregs, potentially promoting colonization, propagation, and invasion of S. aureus in the host.

Transposase-Mediated Excision, Conjugative Transfer, and Diversity of ICE6013 Elements in Staphylococcus aureus.

ICE6013 represents one of two families of integrative conjugative elements (ICEs) identified in the pan-genome of the human and animal pathogen Staphylococcus aureus Here we investigated the excision and conjugation functions of ICE6013 and further characterized the diversity of this element. ICE6013 excision was not significantly affected by growth, temperature, pH, or UV exposure and did not depend on recA The IS30-like DDE transposase (Tpase; encoded by orf1 and orf2) of ICE6013 must be uninterrupted for excision to occur, whereas disrupting three of the other open reading frames (ORFs) on the element significantly affects the level of excision. We demonstrate that ICE6013 conjugatively transfers to different S. aureus backgrounds at frequencies approaching that of the conjugative plasmid pGO1. We found that excision is required for conjugation, that not all S. aureus backgrounds are successful recipients, and that transconjugants acquire the ability to transfer ICE6013 Sequencing of chromosomal integration sites in serially passaged transconjugants revealed a significant integration site preference for a 15-bp AT-rich palindromic consensus sequence, which surrounds the 3-bp target site that is duplicated upon integration. A sequence analysis of ICE6013 from different host strains of S. aureus and from eight other species of staphylococci identified seven divergent subfamilies of ICE6013 that include sequences previously classified as a transposon, a plasmid, and various ICEs. In summary, these results indicate that the IS30-like Tpase functions as the ICE6013 recombinase and that ICE6013 represents a diverse family of mobile genetic elements that mediate conjugation in staphylococci.IMPORTANCE Integrative conjugative elements (ICEs) encode the abilities to integrate into and excise from bacterial chromosomes and plasmids and mediate conjugation between bacteria. As agents of horizontal gene transfer, ICEs may affect bacterial evolution. ICE6013 represents one of two known families of ICEs in the pathogen Staphylococcus aureus, but its core functions of excision and conjugation are not well studied. Here, we show that ICE6013 depends on its IS30-like DDE transposase for excision, which is unique among ICEs, and we demonstrate the conjugative transfer and integration site preference of ICE6013 A sequence analysis revealed that ICE6013 has diverged into seven subfamilies that are dispersed among staphylococci.

Autophagic myelin destruction by Schwann cells during Wallerian degeneration and segmental demyelination.

As lysosomal hydrolysis has long been suggested to be responsible for myelin clearance after peripheral nerve injury, in this study, we investigated the possible role of autophagolysosome formation in myelin phagocytosis by Schwann cells and its final contribution to nerve regeneration. We found that the canonical formation of autophagolysosomes was induced in demyelinating Schwann cells after injury, and the inhibition of autophagy via Schwann cell-specific knockout of the atg7 gene or pharmacological intervention of lysosomal function caused a significant delay in myelin clearance. However, Schwann cell dedifferentiation, as demonstrated by extracellular signal-regulated kinase activation and c-Jun induction, and redifferentiation were not significantly affected, and thus the entire repair program progressed normally in atg7 knockout mice. Finally, autophagic Schwann cells were also found during segmental demyelination in a mouse model of inflammatory peripheral neuropathy. Together, our findings suggest that autophagy is the self-myelin destruction mechanism of Schwann cells, but mechanistically, it is a process distinct from Schwann cell plasticity for nerve repair.

Grb2-associated binder-1 is required for neuregulin-1-induced peripheral nerve myelination.

Grb2-associated binders (Gabs) are scaffolding proteins implicated in cell signaling via receptor tyrosine kinases including neuregulin-1(NRG1)-ErbB receptor signaling, which is essential for peripheral nerve myelination. Here, we show that the conditional removal of Gab1 from Schwann cells resulted in hypomyelination and abnormal development of Remak bundles. In contrast, hypomyelination was not observed in conventional Gab2 knock-out mice. Tyrosine phosphorylation of Gab1, but not Gab2, in sciatic nerves was upregulated during the myelination period and was found to be suppressed in NRG1-type III(+/-) mice, which display a hypomyelinated phenotype similar to that observed in Gab1 knock-out mice. Gab1 knock-out and NRG1-type III(+/-) mice both exhibited reduced extracellular signal-regulated kinase activity in myelinating nerves. In addition, Krox20, a transcription factor that is critical for myelination, has been identified as a target of the NRG1-Gab1 pathway during the myelination process. Our findings suggest that Gab1 is an essential component of NRG1-type III signaling during peripheral nerve development.

Characterization of a novel two-component regulatory system, HptRS, the regulator for the hexose phosphate transport system in Staphylococcus aureus.

Hexose phosphate is an important carbon source within the cytoplasm of host cells. Bacterial pathogens that invade, survive, and multiply within various host epithelial cells exploit hexose phosphates from the host cytoplasm through the hexose phosphate transport (HPT) system to gain energy and synthesize cellular components. In Escherichia coli, the HPT system consists of a two-component regulatory system (UhpAB) and a phosphate sensor protein (UhpC) that tightly regulate expression of a hexose phosphate transporter (UhpT). Although growing evidence suggests that Staphylococcus aureus also can invade, survive, and multiply within various host epithelial cells, the genetic elements involved in the HPT system in S. aureus have not been characterized yet. In this study, we identified and characterized the HPT system in S. aureus that includes the hptRS (a novel two-component regulatory system), the hptA (a putative phosphate sensor), and the uhpT (a hexose phosphate transporter) genes. The hptA, hptRS, and uhpT markerless deletion mutants were generated by an allelic replacement method using a modified pMAD-CM-GFPuv vector system. We demonstrated that both hptA and hptRS are required to positively regulate transcription of uhpT in response to extracellular phosphates, such as glycerol-3-phosphate (G3P), glucose-6-phosphate (G6P), and fosfomycin. Mutational studies revealed that disruption of the hptA, hptRS, or uhpT gene impaired the growth of bacteria when the available carbon source was limited to G6P, impaired survival/multiplication within various types of host cells, and increased resistance to fosfomycin. The results of this study suggest that the HPT system plays an important role in adaptation of S. aureus within the host cells and could be an important target for developing novel antistaphylococcal therapies.

L-arginine supplementation abrogates hypoxia-induced virulence of Staphylococcus aureus in a murine diabetic pressure wound model.

Diabetic foot ulcers (DFUs) are the most common complications of diabetes resulting from hyperglycemia leading to ischemic hypoxic tissue and nerve damage. Staphylococcus aureus is the most frequently isolated bacteria from DFUs and causes severe necrotic infections leading to amputations with a poor 5-year survival rate. However, very little is known about the mechanisms by which S. aureus dominantly colonizes and causes severe disease in DFUs. Herein, we utilized a pressure wound model in diabetic TALLYHO/JngJ mice to reproduce ischemic hypoxic tissue damage seen in DFUs and demonstrated that anaerobic fermentative growth of S. aureus significantly increased the virulence and the severity of disease by activating two-component regulatory systems leading to expression of virulence factors. Our in vitro studies showed that supplementation of nitrate as a terminal electron acceptor promotes anaerobic respiration and suppresses the expression of S. aureus virulence factors through inactivation of two-component regulatory systems, suggesting potential therapeutic benefits by promoting anaerobic nitrate respiration. Our in vivo studies revealed that dietary supplementation of L-arginine (L-Arg) significantly attenuated the severity of disease caused by S. aureus in the pressure wound model by providing nitrate. Collectively, these findings highlight the importance of anaerobic fermentative growth in S. aureus pathogenesis and the potential of dietary L-Arg supplementation as a therapeutic to prevent severe S. aureus infection in DFUs.IMPORTANCES. aureus is the most common cause of infection in DFUs, often resulting in lower-extremity amputation with a distressingly poor 5-year survival rate. Treatment for S. aureus infections has largely remained unchanged for decades and involves tissue debridement with antibiotic therapy. With high levels of conservative treatment failure, recurrence of ulcers, and antibiotic resistance, a new approach is necessary to prevent lower-extremity amputations. Nutritional aspects of DFU treatment have largely been overlooked as there has been contradictory clinical trial evidence, but very few in vitro and in vivo modelings of nutritional treatment studies have been performed. Here we demonstrate that dietary supplementation of L-Arg in a diabetic mouse model significantly reduced duration and severity of disease caused by S. aureus. These findings suggest that L-Arg supplementation could be useful as a potential preventive measure against severe S. aureus infections in DFUs.

The Neuregulin-Rac-MKK7 pathway regulates antagonistic c-jun/Krox20 expression in Schwann cell dedifferentiation.

Schwann cells respond to nerve injury by dedifferentiating into immature states and producing neurotrophic factors, two actions that facilitate successful regeneration of axons. Previous reports have implicated the Raf-ERK cascade and the expression of c-jun in these Schwann cell responses. Here we used cultured primary Schwann cells to demonstrate that active Rac1 GTPase (Rac) functions as a negative regulator of Schwann cell differentiation by upregulating c-jun and downregulating Krox20 through the MKK7-JNK pathway, but not through the Raf-ERK pathway. The activation of MKK7 and induction of c-jun in sciatic nerves after axotomy was blocked by Rac inhibition. Microarray experiments revealed that the expression of regeneration-associated genes, such as glial cell line-derived neurotrophic factor and p75 neurotrophin receptor, after nerve injury was dependent on Rac but not on ERK. Finally, the inhibition of ErbB2 signaling prevented MKK7 activation, c-jun induction, and Rac-dependent gene expression in sciatic nerve explant cultures. Taken together, our results indicate that the neuregulin-Rac-MKK7-JNK/c-jun pathway regulates Schwann cell dedifferentiation following nerve injury.

A novel core genome-encoded superantigen contributes to lethality of community-associated MRSA necrotizing pneumonia.

Bacterial superantigens (SAg) stimulate T-cell hyper-activation resulting in immune modulation and severe systemic illnesses such as Staphylococcus aureus toxic shock syndrome. However, all known S. aureus SAgs are encoded by mobile genetic elements and are made by only a proportion of strains. Here, we report the discovery of a novel SAg staphylococcal enterotoxin-like toxin X (SElX) encoded in the core genome of 95% of phylogenetically diverse S. aureus strains from human and animal infections, including the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 clone. SElX has a unique predicted structure characterized by a truncated SAg B-domain, but exhibits the characteristic biological activities of a SAg including Vß-specific T-cell mitogenicity, pyrogenicity and endotoxin enhancement. In addition, SElX is expressed by clinical isolates in vitro, and during human, bovine, and ovine infections, consistent with a broad role in S. aureus infections of multiple host species. Phylogenetic analysis suggests that the selx gene was acquired horizontally by a progenitor of the S. aureus species, followed by allelic diversification by point mutation and assortative recombination resulting in at least 17 different alleles among the major pathogenic clones. Of note, SElX variants made by human- or ruminant-specific S. aureus clones demonstrated overlapping but distinct Vß activation profiles for human and bovine lymphocytes, indicating functional diversification of SElX in different host species. Importantly, SElX made by CA-MRSA USA300 contributed to lethality in a rabbit model of necrotizing pneumonia revealing a novel virulence determinant of CA-MRSA disease pathogenesis. Taken together, we report the discovery and characterization of a unique core genome-encoded superantigen, providing new insights into the evolution of pathogenic S. aureus and the molecular basis for severe infections caused by the CA-MRSA USA300 epidemic clone.

Role of a new intimin/invasin-like protein in Yersinia pestis virulence.

A comprehensive TnphoA mutant library was constructed in Yersinia pestis KIM6 to identify surface proteins involved in Y. pestis host cell invasion and bacterial virulence. Insertion site analysis of the library repeatedly identified a 9,042-bp chromosomal gene (YPO3944), intimin/invasin-like protein (Ilp), similar to the Gram-negative intimin/invasin family of surface proteins. Deletion mutants of ilp were generated in Y. pestis strains KIM5(pCD1(+)) Pgm(-) (pigmentation negative)/, KIM6(pCD1(-)) Pgm(+), and CO92. Comparative analyses were done with the deletions and the parental wild type for bacterial adhesion to and internalization by HEp-2 cells in vitro, infectivity and maintenance in the flea vector, and lethality in murine models of systemic and pneumonic plague. Deletion of ilp had no effect on bacterial blockage of flea blood feeding or colonization. The Y. pestis KIM5 Δilp strain had reduced adhesion to and internalization by HEp-2 cells compared to the parental wild-type strain (P < 0.05). Following intravenous challenge with Y. pestis KIM5 Δilp, mice had a delayed time to death and reduced dissemination to the lungs, livers, and kidneys as monitored by in vivo imaging using a lux reporter system (in vivo imaging system [IVIS]) and bacterial counts. Intranasal challenge in mice with Y. pestis CO92 Δilp had a 55-fold increase in the 50% lethal dose ([LD(50)] 1.64 × 10(4) CFU) compared to the parental wild-type strain LD(50) (2.98 × 10(2) CFU). These findings identified Ilp as a novel virulence factor of Y. pestis.

Local production of serum amyloid a is implicated in the induction of macrophage chemoattractants in Schwann cells during wallerian degeneration of peripheral nerves.

The elevation of serum levels of serum amyloid A (SAA) has been regarded as an acute reactive response following inflammation and various types of injuries. SAA from the liver and extrahepatic tissues plays an immunomodulatory role in a variety of pathophysiological conditions. Inflammatory cytokines in the peripheral nerves have been implicated in the Wallerian degeneration of peripheral nerves after injury and in certain types of inflammatory neuropathies. In the present study, we found that a sciatic nerve axotomy could induce an increase of SAA1 and SAA3 mRNA expression in sciatic nerves. Immunohistochemical staining showed that Schwann cells are the primary sources of SAA production after nerve injury. In addition, interleukin-6-null mice, but not tumor necrosis factor-α-null mice showed a defect in the production of SAA1 in sciatic nerve following injury. Dexamethasone treatment enhanced the expression and secretion of SAA1 and SAA3 in sciatic nerve explants cultures, suggesting that interleukin-6 and corticosteroids might be major regulators for SAA production in Schwann cells following injury. Moreover, the stimulation of Schwann cells with SAA1 elicited the production of the macrophage chemoattractants, Ccl2 and Ccl3, in part through a G-protein coupled receptor. Our findings suggest that locally produced SAA might play an important role in Wallerian degeneration after peripheral nerve injury.

The Group B Streptococcal Adhesin BspC Interacts with Host Cytokeratin 19 To Promote Colonization of the Female Reproductive Tract.

Streptococcus agalactiae, otherwise known as Group B Streptococcus (GBS), is an opportunistic pathogen that vaginally colonizes approximately one third of healthy women. During pregnancy, this can lead to in utero infection, resulting in premature rupture of membranes, chorioamnionitis, and stillbirths. Furthermore, GBS causes serious infection in newborns, including sepsis, pneumonia, and meningitis. Previous studies have indicated that GBS antigen (Ag) I/II family proteins promote interaction with vaginal epithelial cells; thus, we hypothesized that the Ag I/II Group B streptococcal surface protein C (BspC) contributes to GBS colonization of the female reproductive tract (FRT). Here, we show that a ΔbspC mutant has decreased bacterial adherence to vaginal, ecto-, and endocervical cells, as well as decreased auto-aggregation and biofilm-like formation on cell monolayers. Using a murine model of vaginal colonization, we observed that the ΔbspC mutant strain exhibited a significant fitness defect compared to wild-type (WT) GBS and was less able to ascend to the cervix and uterus in vivo, resulting in reduced neutrophil chemokine signaling. Furthermore, we determined that BspC interacts directly with the host intermediate filament protein cytokeratin 19 (K19). Surface localization of K19 was increased during GBS infection, and interaction was mediated by the BspC variable (V) domain. Finally, mice treated with a drug that targets the BspC V-domain exhibited reduced bacterial loads in the vaginal lumen and reproductive tissues. These results demonstrate the importance of BspC in promoting GBS colonization of the FRT and that it may be targeted therapeutically to reduce GBS vaginal persistence and ascending infection. IMPORTANCE Group B Streptococcus (GBS) asymptomatically colonizes the female reproductive tract (FRT) of up to one third of women, but GBS carriage can lead to adverse pregnancy outcomes, including premature rupture of membranes, preterm labor, and chorioamnionitis. GBS colonization during pregnancy is also the largest predisposing factor for neonatal GBS disease, including pneumonia, sepsis, and meningitis. The molecular interactions between bacterial surface proteins and the host cell receptors that promote GBS colonization are vastly understudied, and a better understanding would facilitate development of novel therapeutics to prevent GBS colonization and disease. Here, we characterize the role of the GBS surface protein BspC in colonization of the FRT. We show for the first time that GBS infection induces cytokeratin 19 (K19) surface localization on vaginal epithelial cells; GBS then uses the BspC V-domain to interact with K19 to promote colonization and ascending infection. Furthermore, this interaction can be targeted therapeutically to reduce GBS carriage.

An Enterotoxin-Bearing Pathogenicity Island in Staphylococcus epidermidis.

Cocolonization of human mucosal surfaces causes frequent encounters between various staphylococcal species, creating opportunities for the horizontal acquisition of mobile genetic elements. The majority of Staphylococcus aureus toxins and virulence factors are encoded on S. aureus pathogenicity islands (SaPIs). Horizontal movement of SaPIs between S. aureus strains plays a role in the evolution of virulent clinical isolates. Although there have been reports of the production of toxic shock syndrome toxin 1 (TSST-1), enterotoxin, and other superantigens by coagulase-negative staphylococci, no associated pathogenicity islands have been found in the genome of Staphylococcus epidermidis, a generally less virulent relative of S. aureus. We show here the first evidence of a composite S. epidermidis pathogenicity island (SePI), the product of multiple insertions in the genome of a clinical isolate. The taxonomic placement of S. epidermidis strain FRI909 was confirmed by a number of biochemical tests and multilocus sequence typing. The genome sequence of this strain was analyzed for other unique gene clusters and their locations. This pathogenicity island encodes and expresses staphylococcal enterotoxin C3 (SEC3) and staphylococcal enterotoxin-like toxin L (SElL), as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and immunoblotting. We present here an initial characterization of this novel pathogenicity island, and we establish that it is stable, expresses enterotoxins, and is not obviously transmissible by phage transduction. We also describe the genome sequence, excision, replication, and packaging of a novel bacteriophage in S. epidermidis FRI909, as well as attempts to mobilize the SePI element by this phage.

Responses to chemical cross-talk between the Mycobacterium ulcerans toxin, mycolactone, and Staphylococcus aureus.

Buruli ulcer is a neglected tropical disease caused by the environmental pathogen, Mycobacterium ulcerans whose major virulence factor is mycolactone, a lipid cytotoxic molecule. Buruli ulcer has high morbidity, particularly in rural West Africa where the disease is endemic. Data have shown that infected lesions of Buruli ulcer patients can be colonized by quorum sensing bacteria such as Staphylococcus aureus, S. epidermidis, and Pseudomonas aeruginosa, but without typical pathology associated with those pathogens' colonization. M. ulcerans pathogenesis may not only be an individual act but may also be dependent on synergistic or antagonistic mechanisms within a polymicrobial network. Furthermore, co-colonization by these pathogens may promote delayed wound healing, especially after the initiation of antibiotic therapy. Hence, it is important to understand the interaction of M. ulcerans with other bacteria encountered during skin infection. We added mycolactone to S. aureus and incubated for 3, 6 and 24 h. At each timepoint, S. aureus growth and hemolytic activity was measured, and RNA was isolated to measure virulence gene expression through qPCR and RNASeq analyses. Results showed that mycolactone reduced S. aureus hemolytic activity, suppressed hla promoter activity, and attenuated virulence genes, but did not affect S. aureus growth. RNASeq data showed mycolactone greatly impacted S. aureus metabolism. These data are relevant and significant as mycolactone and S. aureus sensing and response at the transcriptional, translational and regulation levels will provide insight into biological mechanisms of interspecific interactions that may play a role in regulation of responses such as effects between M. ulcerans, mycolactone, and S. aureus virulence that will be useful for treatment and prevention.

Pneumococcal surface adhesion A protein (PsaA) interacts with human Annexin A2 on airway epithelial cells.

Streptococcus pneumoniae (pneumococcus) is a normal colonizer of the human nasopharynx capable of causing serious invasive disease. Since colonization of the nasopharynx is a prerequisite for progression to invasive diseases, the development of future protein-based vaccines requires an understanding of the intimate interaction of bacterial adhesins with host receptors. In this study, we identified that pneumococcal surface adhesin A (PsaA), a highly conserved pneumococcal protein known to play an important role in colonization of pneumococcus, can interact with Annexin A2 (ANXA2) on Detroit 562 nasopharyngeal epithelial cells. Lentiviral expression of ANXA2 in HEK 293 T/17 cells, which normally express minimal ANXA2, significantly increased pneumococcal adhesion. Blocking of ANXA2 with recombinant PsaA negatively impacted pneumococcal adherence to ANXA2-transduced HEK cells. These results suggest that ANXA2 is an important host cellular receptor for pneumococcal colonization.

Understanding How Staphylococcal Autolysin Domains Interact With Polystyrene Surfaces.

Biofilms, when formed on medical devices, can cause malfunctions and reduce the efficiency of these devices, thus complicating treatments and serving as a source of infection. The autolysin protein of Staphylococcus epidermidis contributes to its biofilm forming ability, especially on polystyrene surfaces. R2ab and amidase are autolysin protein domains thought to have high affinity to polystyrene surfaces, and they are involved in initial bacterial attachment in S. epidermidis biofilm formation. However, the structural details of R2ab and amidase binding to surfaces are poorly understood. In this study, we have investigated how R2ab and amidase influence biofilm formation on polystyrene surfaces. We have also studied how these proteins interact with polystyrene nanoparticles (PSNPs) using biophysical techniques. Pretreating polystyrene plates with R2ab and amidase domains inhibits biofilm growth relative to a control protein, indicating that these domains bind tightly to polystyrene surfaces and can block bacterial attachment. Correspondingly, we find that both domains interact strongly with anionic, carboxylate-functionalized as well as neutral, non-functionalized PSNPs, suggesting a similar binding interaction for nanoparticles and macroscopic surfaces. Both anionic and neutral PSNPs induce changes to the secondary structure of both R2ab and amidase as monitored by circular dichroism (CD) spectroscopy. These changes are very similar, though not identical, for both types of PSNPs, suggesting that carboxylate functionalization is only a small perturbation for R2ab and amidase binding. This structural change is also seen in limited proteolysis experiments, which exhibit substantial differences for both proteins when in the presence of carboxylate PSNPs. Overall, our results demonstrate that the R2ab and amidase domains strongly favor adsorption to polystyrene surfaces, and that surface adsorption destabilizes the secondary structure of these domains. Bacterial attachment to polystyrene surfaces during the initial phases of biofilm formation, therefore, may be mediated by aromatic residues, since these residues are known to drive adsorption to PSNPs. Together, these experiments can be used to develop new strategies for biofilm eradication, ensuring the proper long-lived functioning of medical devices.

Role of Glucose-6-Phosphate in Metabolic Adaptation of Staphylococcus aureus in Diabetes.

Diabetic foot ulcer (DFU) is the most common and costly sequela of diabetes mellitus, often leading to lower-extremity amputation with poor 5-year survival rates. Staphylococcus aureus is the most prevalent pathogen isolated from DFU, suggesting adaptation of S. aureus to the unique metabolic conditions of diabetes. Diabetes is a complex metabolic disorder with increases not only in serum glucose levels but also in levels of other sugars, including fructose, mannose, and glucose-6-phosphate (G6P). However, the effect of metabolism of these sugars on the pathogenesis of S. aureus is not fully understood. In this study, we demonstrated that metabolism of G6P, fructose, and mannose induced greater expression of staphylococcal virulence factors than did glucose metabolism, but only G6P effects were independent of glucose-mediated carbon catabolite repression, suggesting a physiologically relevant role in diabetes. Our in vivo studies further demonstrated that G6P was highly present in skin adipose tissues of diabetic TALLYHO/JngJ mice, and subcutaneous infection with S. aureus caused significantly greater tissue necrosis and bacterial burden, compared to nondiabetic SWR/J mice. Finally, enhanced pathogenesis of S. aureus in diabetic TALLYHO/JngJ mice was significantly attenuated by deletion of the hexose phosphate transport (HPT) system. These results suggest that G6P is an important metabolic signal for S. aureus, enhancing the virulence in diabetes. A better understanding of how G6P metabolism is linked to the virulence of S. aureus will lead to the development of novel alternative therapeutics. IMPORTANCE Sugars are essential nutrients for S. aureus to survive and proliferate within the host. Because elevated serum glucose levels are a hallmark of diabetes, most studies have focused on the effect of glucose metabolism, and very little is known regarding the effects of metabolism of other sugars on the pathogenesis of S. aureus in diabetes. In this study, we demonstrated that G6P, which is highly present in diabetes, can induce expression of staphylococcal virulence factors that cause severe tissue necrosis and bacterial burden in skin infections. Our results highlight the importance of nutritional control of blood sugar levels, not only glucose but also other highly metabolizable sugars such as G6P. A better understanding of how activation of the HPT system is linked to the virulence of S. aureus will guide development of novel alternative therapeutics.

A quantitative real time PCR method to analyze T cell receptor Vbeta subgroup expansion by staphylococcal superantigens.

BACKGROUND: Staphylococcal enterotoxins (SEs), SE-like (SEl) toxins, and toxic shock syndrome toxin-1 (TSST-1), produced by Staphylococcus aureus, belong to the subgroup of microbial superantigens (SAgs). SAgs induce clonal proliferation of T cells bearing specific variable regions of the T cell receptor beta chain (Vbeta). Quantitative real time PCR (qRT-PCR) has become widely accepted for rapid and reproducible mRNA quantification. Although the quantification of Vbeta subgroups using qRT-PCR has been reported, qRT-PCR using both primers annealing to selected Vbeta nucleotide sequences and SYBR Green I reporter has not been applied to assess Vbeta-dependent expansion of T cells by SAgs. METHODS: Human peripheral blood mononuclear cells were stimulated with various SAgs or a monoclonal antibody specific to human CD3. Highly specific expansion of Vbeta subgroups was assessed by qRT-PCR using SYBR Green I reporter and primers corresponding to selected Vbeta nucleotide sequences. RESULTS: qRT-PCR specificities were confirmed by sequencing amplified PCR products and melting curve analysis. To assess qRT-PCR efficiencies, standard curves were generated for each primer set. The average slope and R2 of standard curves were -3.3764 +/- 0.0245 and 0.99856 +/- 0.000478, respectively, demonstrating that the qRT-PCR established in this study is highly efficient. With some exceptions, SAg Vbeta specificities observed in this study were similar to those reported in previous studies. CONCLUSIONS: The qRT-PCR method established in this study produced an accurate and reproducible assessment of Vbeta-dependent expansion of human T cells by staphylococcal SAgs. This method could be a useful tool in the characterization T cell proliferation by newly discovered SAg and in the investigation of biological effects of SAgs linked to pathogenesis.

Population Analysis of Staphylococcus aureus Reveals a Cryptic, Highly Prevalent Superantigen SElW That Contributes to the Pathogenesis of Bacteremia.

Staphylococcal superantigens (SAgs) are a family of secreted toxins that stimulate T cell activation and are associated with an array of diseases in humans and livestock. Most SAgs produced by Staphylococcus aureus are encoded by mobile genetic elements, such as pathogenicity islands, bacteriophages, and plasmids, in a strain-dependent manner. Here, we carried out a population genomic analysis of >800 staphylococcal isolates representing the breadth of S. aureus diversity to investigate the distribution of all 26 identified SAg genes. Up to 14 SAg genes were identified per isolate with the most common gene selw (encoding a putative SAg, SElW) identified in 97% of isolates. Most isolates (62.5%) have a full-length open reading frame of selw with an alternative TTG start codon that may have precluded functional characterization of SElW to date. Here, we demonstrate that S. aureus uses the TTG start codon to translate a potent SAg SElW that induces Vß-specific T cell proliferation, a defining feature of classical SAgs. SElW is the only SAg predicted to be expressed by isolates of the CC398 lineage, an important human and livestock epidemic clone. Deletion of selw in a representative CC398 clinical isolate, S. aureus NM001, resulted in complete loss of T cell mitogenicity in vitro, and in vivo expression of SElW by S. aureus increased the bacterial load in the liver during bloodstream infection of SAg-sensitive HLA-DR4 transgenic mice. Overall, we report the characterization of a novel, highly prevalent, and potent SAg that contributes to the pathogenesis of S. aureus infection.IMPORTANCEStaphylococcus aureus is an important human and animal pathogen associated with an array of diseases, including life-threatening necrotizing pneumonia and infective endocarditis. The success of S. aureus as a pathogen has been linked in part to its ability to manipulate the host immune response through the secretion of toxins and immune evasion molecules. The staphylococcal superantigens (SAgs) have been studied for decades, but their role in S. aureus pathogenesis is not well understood, and an appreciation for how SAgs manipulate the host immune response to promote infection may be crucial for the development of novel intervention strategies. Here, we characterized a widely prevalent, previously cryptic, staphylococcal SAg, SElW, that contributes to the severity of S. aureus infections caused by an important epidemic clone of S. aureus CC398. Our findings add to the understanding of staphylococcal SAg diversity and function and provide new insights into the capacity of S. aureus to cause disease.