Peptidoglycan Contribution to the B Cell Superantigen Activity of Staphylococcal Protein A.

Staphylococcus aureus causes reiterative and chronic persistent infections. This can be explained by the formidable ability of this pathogen to escape immune surveillance mechanisms. Cells of S. aureus display the abundant staphylococcal protein A (SpA). SpA binds to immunoglobulin (Ig) molecules and coats the bacterial surface to prevent phagocytic uptake. SpA also binds and cross-links variable heavy 3 (VH3) idiotype (IgM) B cell receptors, promoting B cell expansion and the secretion of nonspecific VH3-IgM via a mechanism requiring CD4+ T cell help. SpA binding to antibodies is mediated by the N-terminal Ig-binding domains (IgBDs). The so-called region X, uncharacterized LysM domain, and C-terminal LPXTG sorting signal for peptidoglycan attachment complete the linear structure of the protein. Here, we report that both the LysM domain and the LPXTG motif sorting signal are required for the B cell superantigen activity of SpA in a mouse model of infection. SpA molecules purified from staphylococcal cultures are sufficient to exert B cell superantigen activity and promote immunoglobulin secretion as long as they carry intact LysM and LPXTG motif domains with bound peptidoglycan fragments. The LysM domain binds the glycan chains of peptidoglycan fragments, whereas the LPXTG motif is covalently linked to wall peptides lacking glycan. These findings emphasize the complexity of SpA interactions with B cell receptors.IMPORTANCE The LysM domain is found in all kingdoms of life. While their function in mammals is not known, LysM domains of bacteria and their phage parasites are associated with enzymes that cleave or remodel peptidoglycan. Plants recognize microbe-associated molecular patterns such as chitin via receptors endowed with LysM-containing ectodomains. In plants, such receptors play equally important roles in defense and symbiosis signaling. SpA of S. aureus carries a LysM domain that binds glycan strands of peptidoglycan to influence defined B cell responses that divert pathogen-specific adaptive immune responses.

Acceptability and Feasibility of Early Palliative Care Among Women with Advanced Epithelial Ovarian Cancer: A Randomized Controlled Pilot Study.

OBJECTIVE: To evaluate the acceptability of early palliative care (EPC) among patients with advanced ovarian cancer and to determine the feasibility of larger-scale phase III trials. METHODS: We performed a randomized controlled pilot study of adult women (>18 years) with pathologically confirmed epithelial ovarian cancer that had recurred or progressed on first-line therapy and had no immediate need for palliative care. We randomly assigned patients to either EPC or standard oncologic care (SOC), and collected patient-reported outcomes (PRO) at baseline, 3 months, and 6 months; end-of-life care quality indicators were collected at study completion. Study endpoints were rates of enrollment, EPC adherence, and PRO completion. RESULTS: Of 32 eligible patients approached, 23 enrolled (72%; 95% CI 53-86) and were randomly assigned to either EPC (n = 12) or SOC (n = 11). At baseline, participants had poor physical and emotional wellbeing, high rates of depression (65%), and understood that their disease was not curable (87%). Eleven patients (92%; 95% CI 62-100) attended their EPC consultation, and all visits took place within 4 weeks of enrollment. However, PRO completion was low due to deaths by 3 (5/23) and 6 months (9/23). CONCLUSION: Patients had accurate perceptions of their disease status, were willing to be randomly assigned to EPC, and attended scheduled appointments. However, a definitive trial in this group is not feasible without major adjustments to eligibility criteria and a multicentre, international effort. We propose that EPC be considered routinely at progression or recurrence given patients' symptom burden and clear acceptance of the intervention, as well as evidence of benefit from adequately powered trials in other malignancies.

Regulated cleavage of glycan strands by the murein hydrolase SagB in S. aureus involves a direct interaction with LyrA (SpdC).

LyrA (SpdC), a homologue of eukaryotic CAAX proteases that act on prenylated substrates, has been implicated in the assembly of several pathways of the envelope of Staphylococcus aureus. We described earlier the Lysostaphin resistance (Lyr) and Staphylococcal protein A display (Spd) phenotypes associated with loss of the lyrA (spdC) gene. However, a direct contribution to the assembly of pentaglycine crossbridges, the target of lysostaphin cleavage in S. aureus peptidoglycan, or of Staphylococcal protein A attachment to peptidoglycan could not be attributed directly to LyrA (SpdC). These two processes are catalyzed by the Fem factors and Sortase A, respectively. To gain insight into the function of LyrA (SpdC), here we use affinity chromatography and LC-MS/MS analysis and report that LyrA interacts with SagB. SagB cleaves glycan strands of peptidoglycan to achieve physiological length. Similar to sagB peptidoglycan, lyrA peptidoglycan contains extended glycan strands. Purified lyrA peptidoglycan can still be cleaved to physiological length by SagB in vitro LyrA does not modify or cleave peptidoglycan, it also does not modify or stabilize SagB. The membrane bound domain of LyrA is sufficient to support SagB activity but predicted 'CAAX enzyme' catalytic residues in this domain are dispensable. We speculate that LyrA exerts its effect on bacterial prenyl substrates, specifically undecaprenol-bound peptidoglycan substrates of SagB, to help control glycan length. Such an activity also explains the Lyr and Spd phenotypes observed earlier.IMPORTANCE Peptidoglycan is assembled on the trans side of the plasma membrane from lipid II precursors into glycan chains that are crosslinked at stem peptides. In S. aureus, SagB, a membrane-associated N-acetylglucosaminidase, cleaves polymerized glycan chains to their physiological length. Deletion of sagB is associated with longer glycan strands in peptidoglycan, altered protein trafficking and secretion in the envelope, and aberrant excretion of cytosolic proteins. It is not clear whether SagB, with its single transmembrane segment, serves as the molecular ruler of glycan chains or whether other factors modulate its activity. Here, we show that LyrA (SpdC), a protein of the CAAX type II prenyl endopeptidase family, modulates SagB activity via interaction though its transmembrane domain.

FmhA and FmhC of Staphylococcus aureus incorporate serine residues into peptidoglycan cross-bridges.

Staphylococcal peptidoglycan is characterized by pentaglycine cross-bridges that are cross-linked between adjacent wall peptides by penicillin-binding proteins to confer robustness and flexibility. In Staphylococcus aureus, pentaglycine cross-bridges are synthesized by three proteins: FemX adds the first glycine, and the homodimers FemA and FemB sequentially add two Gly-Gly dipeptides. Occasionally, serine residues are also incorporated into the cross-bridges by enzymes that have heretofore not been identified. Here, we show that the FemA/FemB homologues FmhA and FmhC pair with FemA and FemB to incorporate Gly-Ser dipeptides into cross-bridges and to confer resistance to lysostaphin, a secreted bacteriocin that cleaves the pentaglycine cross-bridge. FmhA incorporates serine residues at positions 3 and 5 of the cross-bridge. In contrast, FmhC incorporates a single serine at position 5. Serine incorporation also lowers resistance toward oxacillin, an antibiotic that targets penicillin-binding proteins, in both methicillin-sensitive and methicillin-resistant strains of S. aureus FmhC is encoded by a gene immediately adjacent to lytN, which specifies a hydrolase that cleaves the bond between the fifth glycine of cross-bridges and the alanine of the adjacent stem peptide. In this manner, LytN facilitates the separation of daughter cells. Cell wall damage induced upon lytN overexpression can be alleviated by overexpression of fmhC. Together, these observations suggest that FmhA and FmhC generate peptidoglycan cross-bridges with unique serine patterns that provide protection from endogenous murein hydrolases governing cell division and from bacteriocins produced by microbial competitors.

EssH Peptidoglycan Hydrolase Enables Staphylococcus aureus Type VII Secretion across the Bacterial Cell Wall Envelope.

The ESAT-6-like secretion system (ESS) of Staphylococcus aureus is assembled in the bacterial membrane from core components that promote the secretion of WXG-like proteins (EsxA, EsxB, EsxC, and EsxD) and the EssD effector. Genes encoding the ESS secretion machinery components, effector, and WXG-like proteins are located in the ess locus. Here, we identify essH, a heretofore uncharacterized gene of the ess locus, whose product is secreted via an N-terminal signal peptide into the extracellular medium of staphylococcal cultures. EssH exhibits two peptidoglycan hydrolase activities, cleaving the pentaglycine cross bridge and the amide bond of N-acetylmuramyl-l-alanine, thereby separating glycan chains and wall peptides with cleaved cross bridges. Unlike other peptidoglycan hydrolases, EssH does not promote the lysis of staphylococci. EssH residues Cys199 and His254, which are conserved in other CHAP domain enzymes, are required for peptidoglycan hydrolase activity and for S. aureus ESS secretion. These data suggest that EssH and its murein hydrolase activity are required for protein secretion by the ESS pathway.IMPORTANCE Gene clusters encoding WXG-like proteins and FtsK/SpoIIIE-like P loop ATPases in Firmicutes encode type 7b secretion systems (T7bSS) for the transport of select protein substrates. The Staphylococcus aureus T7bSS assembles in the bacterial membrane and promotes the secretion of WXG-like proteins and effectors. The mechanisms whereby staphylococci extend the T7SS across the bacterial cell wall envelope are not known. Here, we show that staphylococci secrete EssH to cleave their peptidoglycan, thereby enabling T7bSS transport of proteins across the bacterial cell wall envelope.

Inducible Expression of spo0A as a Universal Tool for Studying Sporulation in Clostridium difficile.

Clostridium difficile remains a leading nosocomial pathogen, putting considerable strain on the healthcare system. The ability to form endospores, highly resistant to environmental insults, is key to its persistence and transmission. However, important differences exist between the sporulation pathways of C. difficile and the model Gram-positive organism Bacillus subtilis. Amongst the challenges in studying sporulation in C. difficile is the relatively poor levels of sporulation and high heterogeneity in the sporulation process. To overcome these limitations we placed Ptet regulatory elements upstream of the master regulator of sporulation, spo0A, generating a new strain that can be artificially induced to sporulate by addition of anhydrotetracycline (ATc). We demonstrate that this strain is asporogenous in the absence of ATc, and that ATc can be used to drive faster and more efficient sporulation. Induction of Spo0A is titratable and this can be used in the study of the spo0A regulon both in vitro and in vivo, as demonstrated using a mouse model of C. difficile infection (CDI). Insights into differences between the sporulation pathways in B. subtilis and C. difficile gained by study of the inducible strain are discussed, further highlighting the universal interest of this tool. The Ptet-spo0A strain provides a useful background in which to generate mutations in genes involved in sporulation, therefore providing an exciting new tool to unravel key aspects of sporulation in C. difficile.

Increased toxin expression in a Clostridium difficile mfd mutant.

BACKGROUND: The symptoms of Clostridium difficile infection are mediated primarily by two toxins, TcdA and TcdB, the expression of which is governed by a multitude of factors including nutrient availability, growth phase and cell stress. Several global regulators have been implicated in the regulation of toxin expression, such as CcpA and CodY. RESULTS: During attempts to insertionally inactivate a putative secondary cell wall polysaccharide synthesis gene, we obtained several mutants containing off-target insertions. One mutant displayed an unusual branched colony morphology and was investigated further. Marker recovery revealed an insertion in mfd, a gene encoding a transcription-coupled repair factor. The mfd mutant exhibited pleiotropic effects, in particular increased expression of both toxin A and B (TcdA and TcdB) compared to the parental strain. Western blotting and cellular cytotoxicity assays revealed increased expression across all time points over a 24 h period, with inactivation of mfd resulting in at least a 10 fold increase in cell cytotoxicity. qRT-PCR demonstrated the upregulation of both toxins occurred on a transcriptional level. All effects of the mfd mutation were complemented by a plasmid-encoded copy of mfd, showing the effects are not due to polar effects of the intron insertion or to second site mutations. CONCLUSIONS: This study adds Mfd to the repertoire of factors involved in regulation of toxin expression in Clostridium difficile. Mfd is known to remove RNA polymerase molecules from transcriptional sites where it has stalled due to repressor action, preventing transcriptional read through. The consistently high levels of toxin in the C. difficile mfd mutant indicate this process is inefficient leading to transcriptional de-repression.

Clostridium difficile surface proteins are anchored to the cell wall using CWB2 motifs that recognise the anionic polymer PSII.

Gram-positive surface proteins can be covalently or non-covalently anchored to the cell wall and can impart important properties on the bacterium in respect of cell envelope organisation and interaction with the environment. We describe here a mechanism of protein anchoring involving tandem CWB2 motifs found in a large number of cell wall proteins in the Firmicutes. In the Clostridium difficile cell wall protein family, we show the three tandem repeats of the CWB2 motif are essential for correct anchoring to the cell wall. CWB2 repeats are non-identical and cannot substitute for each other, as shown by the secretion into the culture supernatant of proteins containing variations in the patterns of repeats. A conserved Ile Leu Leu sequence within the CWB2 repeats is essential for correct anchoring, although a preceding proline residue is dispensable. We propose a likely genetic locus encoding synthesis of the anionic polymer PSII and, using RNA knock-down of key genes, reveal subtle effects on cell wall composition. We show that the anionic polymer PSII binds two cell wall proteins, SlpA and Cwp2, and these interactions require the CWB2 repeats, defining a new mechanism of protein anchoring in Gram-positive bacteria.

Sleep architecture in ziprasidone-treated bipolar depression: a pilot study.

OBJECTIVES: This study investigated the effect of ziprasidone augmentation therapy on sleep architecture in bipolar depression. METHODS: We conducted a double-blind, randomized, placebo-controlled clinical pilot trial of ziprasidone versus placebo in Diagnostic and Statistical Manual of Mental Disorders, fourth edition bipolar disorder with current major depressive episode. The effects during acute (2-5 days) and continuation treatment (28-31 days) were measured. Main outcomes were sleep architecture variables including rapid eye movement sleep (REM) and slow wave sleep (SWS) measured by polysomnography. Secondary outcomes included subjective sleep quality measures and illness severity measures including the 17-item Hamilton Depression Rating Scale (HAMD-17), Montgomery Asberg Depression Rating Scale (MADRS), Hamilton Anxiety Rating Scale (HAMA) and Clinical Global Illness Severity (CGI-S) scores. RESULTS: The completer analysis comprised of 14 patients (ziprasidone, N = 8 and placebo, N = 6). Latency to REM, duration of SWS, duration of stage 2 sleep, total sleep time, onset to sleep latency, number of awakenings and overall sleep efficiency significantly improved in ziprasidone-treated participants over placebo. CGI-S and HAMA scores also significantly improved. No significant difference between treatment groups was seen on the HAMD-17, MADRS or in self-reported sleep quality. Increase in SWS duration significantly correlated with improvement in CGI-S, however, this finding did not withstand Bonferroni correction. CONCLUSION: Adjunctive ziprasidone treatment alters sleep architecture in patients with bipolar depression, which may partially explain its mechanism of action and merits further investigation.

Do children with central venous line (CVL) dysfunction have increased risk of symptomatic thromboembolism compared to those without CVL-dysfunction, while on cancer therapy?

BACKGROUND: Thromboembolism (TE) and infection are two common complications of central venous line (CVL). Thrombotic CVL-dysfunction is a common, yet less studied, complication of CVL. Two retrospective studies have reported significant association of CVL-dysfunction and TE. Recent studies indicate association of CVL-related small clot with infection. Infection is the most common cause of non-cancer related mortality in children with cancer. We and others have shown reduced overall survival (OS) in children with cancer and CVL-dysfunction compared to those without CVL-dysfunction. Despite these observations, to date there are no prospective studies to evaluate the clinical significance of CVL-dysfunction and it's impact on the development of TE, infection, or outcome of children with cancer. STUDY DESIGN: This is a prospective, analytical cohort study conducted at five tertiary care pediatric oncology centers in Ontario. Children (≤ 18 years of age) with non-central nervous system cancers and CVL will be eligible for the study. Primary outcome measure is symptomatic TE and secondary outcomes are infection, recurrence of cancer and death due to any cause. Data will be analyzed using regression analyses. DISCUSSION: The overall objective is to delineate the relationship between CVL-dysfunction, infection and TE. The primary aim is to evaluate the role of CVL-dysfunction as a predictor of symptomatic TE in children with cancer. We hypothesize that children with CVL-dysfunction have activation of the coagulation system resulting in an increased risk of symptomatic TE. The secondary aims are to study the impact of CVL-dysfunction on the rate of infection and the survival [OS and event free survival (EFS)] of children with cancer. We postulate that patients with CVL-dysfunction have an occult CVL-related clot which acts as a microbial focus with resultant increased risk of infection. Further, CVL-dysfunction by itself or in combination with associated complications may cause therapy delays resulting in adverse outcome.This study will help to identify children at high risk for TE and infection. Based on the study results, we will design randomized controlled trials of prophylactic anticoagulant therapy to reduce the incidence of TE and infection. This in turn will help to improve the outcome in children with cancer.

Roles of cysteine proteases Cwp84 and Cwp13 in biogenesis of the cell wall of Clostridium difficile.

Clostridium difficile expresses a number of cell wall proteins, including the abundant high-molecular-weight and low-molecular-weight S-layer proteins (SLPs). These proteins are generated by posttranslational cleavage of the precursor SlpA by the cysteine protease Cwp84. We compared the phenotypes of C. difficile strains containing insertional mutations in either cwp84 or its paralog cwp13 and complemented with plasmids expressing wild-type or mutant forms of their genes. We show that the presence of uncleaved SlpA in the cell wall of the cwp84 mutant results in aberrant retention of other cell wall proteins at the cell surface, as demonstrated by secretion of the proteins Cwp66 and Cwp2 into the growth medium. These phenotypes are restored by complementation with a plasmid expressing wild-type Cwp84 enzyme but not with one encoding a Cys116Ala substitution in the active site. The cwp13 mutant cleaved the SlpA precursor normally and had a wild-type-like colony phenotype. Both Cwp84 and Cwp13 are produced as proenzymes which are processed by cleavage to produce mature enzymes. In the case of Cwp84, this cleavage does not appear to be autocatalytic, whereas in Cwp13 autocatalysis was demonstrated as a Cys109Ala mutant did not undergo processing. Cwp13 appears to have a role in processing of Cwp84 but is not essential for Cwp84 activity. Cwp13 cleaves SlpA in the HMW SLP domain, which we suggest may reflect a role in cleavage and degradation of misfolded proteins at the cell surface.

The acute impact of continuous positive airway pressure on nasal resistance: a randomized controlled comparison.

Subjective nasal obstruction is common among users of continuous positive airway pressure (CPAP). The aim of this study was to measure the acute effect of CPAP on nasal resistance and nasal symptoms in awake normal subjects. Twenty-four healthy CPAP-naive adults [8 men, 16 women; mean age 30 yr (SD 14)] underwent a randomized controlled crossover study comparing nasal CPAP (8 cmH(2)O) for 6 h on one occasion and the control condition (nasal mask without CPAP) on the other. Nasal resistance measurements (posterior active rhinometry) before and after the test exposure were similar on both test days. Nasal resistance during CPAP exposure [2.04 cmH(2)O.l(-1).s (SD 0.72)] was significantly lower than that of the control [2.67 cmH(2)O.l(-1).s (SD 1.07)]: mean difference 0.66 cmH(2)O.l(-1).s, 95% confidence interval 0.19-1.13 cmH(2)O.l(-1).s. The gradient in pressure from CPAP mask to posterior naris during CPAP exposure varied from 1.6 to 2 cmH(2)O but was not significantly different between time points. Subjective nasal symptom scores and peak nasal inspiratory flow rates did not change significantly on either test day. We conclude that in awake CPAP-naive normal subjects, acute CPAP exposure is associated with a reduction in nasal resistance compared with the control condition, but it is not associated with an immediate post-CPAP change in subjective or objective nasal resistance.