Living with an imperfect cell wall: compensation of femAB inactivation in Staphylococcus aureus

Background: Synthesis of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide bridge is catalyzed by the nonribosomal peptidyl transferases FemX, FemA and FemB. Inactivation of the femAB operon reduces the interpeptide to a monoglycine, leading to a poorly crosslinked peptidoglycan. femAB mutants show a reduced growth rate and are hypersusceptible to virtually all antibiotics, including methicillin, making FemAB a potential target to restore β-lactam susceptibility in methicillin-resistant S. aureus (MRSA). Cis-complementation with wild type femAB only restores synthesis of the pentaglycine interpeptide and methicillin resistance, but the growth rate remains low. This study characterizes the adaptations that ensured survival of the cells after femAB inactivation. Results: In addition to slow growth, the cis-complemented femAB mutant showed temperature sensitivity and a higher methicillin resistance than the wild type. Transcriptional profiling paired with reporter metabolite analysis revealed multiple changes in the global transcriptome. A number of transporters for sugars, glycerol, and glycine betaine, some of which could serve as osmoprotectants, were upregulated. Striking differences were found in the transcription of several genes involved in nitrogen metabolism and the arginine-deiminase pathway, an alternative for ATP production. In addition, microarray data indicated enhanced expression of virulence factors that correlated with premature expression of the global regulators sae, sarA, and agr. Conclusion: Survival under conditions preventing normal cell wall formation triggered complex adaptations that incurred a fitness cost, showing the remarkable flexibility of S. aureus to circumvent cell wall damage. Potential FemAB inhibitors would have to be used in combination with other antibiotics to prevent selection of resistant survivors

Antibiotic-resistant bacteria, antibiotic resistance genes, and antibiotic residues in wastewater from a poultry slaughterhouse after conventional and advanced treatments

Slaughterhouse wastewater is considered a reservoir for antibiotic-resistant bacteria and antibiotic residues, which are not sufficiently removed by conventional treatment processes. This study focuses on the occurrence of ESKAPE bacteria (Enterococcus spp., S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, Enterobacter spp.), ESBL (extended-spectrum β-lactamase)-producing E. coli, antibiotic resistance genes (ARGs) and antibiotic residues in wastewater from a poultry slaughterhouse. The efficacy of conventional and advanced treatments (i.e., ozonation) of the in-house wastewater treatment plant regarding their removal was also evaluated. Target culturable bacteria were detected only in the influent and effluent after conventional treatment. High abundances of genes (e.g., bla$_{TEM}$, bla$_{CTX-M-15}$, bla$_{CTX-M-32}$, bla$_{OXA-48}$, bla$_{CMY}$ and mcr-1) of up to 1.48 × 10$^{6}$ copies/100 mL were detected in raw influent. All of them were already significantly reduced by 1–4.2 log units after conventional treatment. Following ozonation, mcr-1 and bla$_{CTX-M-32}$ were further reduced below the limit of detection. Antibiotic residues were detected in 55.6% (n = 10/18) of the wastewater samples. Despite the significant reduction through conventional and advanced treatments, effluents still exhibited high concentrations of some ARGs (e.g., sul1, ermB and bla$_{OXA-48}$), ranging from 1.75 × 10$^{2}$ to 3.44 × 10$^{3}$ copies/100 mL. Thus, a combination of oxidative, adsorptive and membrane-based technologies should be considered

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the \u3e20 distinct compound classes is also reviewed, and commonalities are discussed

What is multidisciplinary cancer care like in practice? a protocol for a mixed-method study to characterise ambulatory oncology services in the Australian public sector

© 2019 Author(s). Introduction An understanding of the real-world provision of oncology outpatient services can help maintain service quality in the face of escalating demand and tight budgets, by informing the design of interventions that improve the effectiveness or efficiency of provision. The aims of this study are threefold. First, to develop an understanding of cancer services in outpatient clinics by characterising the organisation and practice of multidisciplinary care (MDC). Second, to explore the key areas of: (a) clinical decision-making and (b) engagement with patients' supportive needs. Third, to identify barriers to, and facilitators of, the delivery of quality care in these settings. Methods and analysis A suite of mixed-methods studies will be implemented at six hospitals providing cancer outpatient clinics, with a staged roll-out. In Stage One, we will examine policies, use unstructured observations and undertake interviews with key health professionals to characterise the organisation and delivery of MDC. In Stage Two, observations of practice will continue, to deepen our understanding, and to inform two focused studies. The first will explore decision-making practices and the second will examine how staff engage with patients' needs; both studies involve interviews, to complement observation. As part of the study of supportive care, we will examine the implications of an introduction of patient-reported measures (PRMs) into care, adding surveys to interviews before and after PRMs roll-out. Data analysis will account for site-specific and cross-site issues using an adapted Qualitative Rapid Appraisal, Rigorous Analysis approach. Quantitative data from clinician surveys will be statistically analysed and triangulated with the related qualitative study findings. Ethics and dissemination Ethical approval was granted by South Eastern Sydney Local Health District Human Research Ethics Committee (no. 18/207). Findings will be shared with participating hospitals and widely disseminated through publications and presentations

Nucleotide sequence of the lantibiotic Pep5 biosynthetic gene cluster and functional analysis of PepP and PepC. Evidence for a role of PepC in thioether formation

The biosynthesis of Pep5, a lanthionine-containing antimicrobial peptide, is directed by the 20-kbp plasmid pED503. We identified a 7.9-kbp DNA-fragment within this plasmid which covers the information for Pep5 synthesis in the homologous host Staphylococcus epidermidis 5 which has been cured of pED503. This fragment contained, in addition to the previously described structural gene pepA and the immunity gene pepl [Reis, M., Eschbach-Bludau, M., Iglesias-Wind, M. I., Kupke, T. & Sahl, H.-G. (1994) Appl. Env. Microbiol. 60, 2876–2883], a gene pepT coding for a translocator of the ABC transporter family, a gene pepP coding for a serine protease and two genes pepB and pepC coding for putative modification enzymes; the gene arrangement is pepTIAPBC. We analyzed the biosynthetic genes with respect to their function in Pep5 biosynthesis. Deletion of PepT reduced Pep5 production to about 10%, indicating that it can be partially replaced by other host-encoded translocators. Inactivation of PepP by site-directed mutagenesis of the active-site His residue resulted in production of incorrectly processed Pep5 fragments with strongly reduced antimicrobial activity. Deletion of pepB and pepC leads to accumulation of Pep5 prepeptide in the cells without excretion of processed peptide. A pepC-deletion clone did not excrete correctly matured Pep5 but it did produce fragments from which serine and threonine were absent. Only one of these fragments contained a single lanthionine residue out of three expected while the remaining, unmodified cysteine residues could be detected by reaction with Ellman's reagent. These results demonstrate that PepC is a thioether-forming protein and strongly suggest that PepB is responsible for dehydration of serine and threonin

FIP200 claw domain binding to p62 promotes autophagosome formation at ubiquitin condensates

The autophagy cargo receptor p62 facilitates the condensation of misfolded, ubiquitin-positive proteins and their degradation by autophagy, but the molecular mechanism of p62 signaling to the core autophagy machinery is unclear. Here, we show that disordered residues 326-380 of p62 directly interact with the C-terminal region (CTR) of FIP200. Crystal structure determination shows that the FIP200 CTR contains a dimeric globular domain that we designated the "Claw" for its shape. The interaction of p62 with FIP200 is mediated by a positively charged pocket in the Claw, enhanced by p62 phosphorylation, mutually exclusive with the binding of p62 to LC3B, and it promotes degradation of ubiquitinated cargo by autophagy. Furthermore, the recruitment of the FIP200 CTR slows the phase separation of ubiquitinated proteins by p62 in a reconstituted system. Our data provide the molecular basis for a crosstalk between cargo condensation and autophagosome formation

A Staphylococcus aureus ypfP mutant with strongly reduced lipoteichoic acid (LTA) content: LTA governs bacterial surface properties and autolysin activity

Many Gram-positive bacteria produce lipoteichoic acid (LTA) polymers whose physiological roles have remained a matter of debate because of the lack of LTA-deficient mutants. The ypfP gene responsible for biosynthesis of a glycolipid found in LTA was deleted in Staphylococcus aureus SA113, causing 87% reduction of the LTA content. Mass spectrometry and nuclear magnetic resonance spectroscopy revealed that the mutant LTA contained a diacylglycerol anchor instead of the glycolipid, whereas the remaining part was similar to the wild-type polymer except that it was shorter. The LTA mutant strain revealed no major changes in patterns of cell wall proteins or autolytic enzymes compared with the parental strain indicating that LTA may be less important in S. aureus protein attachment than previously thought. However, the autolytic activity of the mutant was strongly reduced demonstrating a role of LTA in controlling autolysin activity. Moreover, the hydrophobicity of the LTA mutant was altered and its ability to form biofilms on plastic was completely abrogated indicating a profound impact of LTA on physicochemical properties of bacterial surfaces. We propose to consider LTA and its biosynthetic enzymes as targets for new antibiofilm strategies

Negative-Ion Photoelectron Spectroscopy, Gas-Phase Acidity, and Thermochemistry of the Peroxyl Radicals CH_3OO and CH_3CH_2OO

Methyl, methyl-d3, and ethyl hydroperoxide anions (CH_3OO-, CD_3OO-, and CH_3CH_2OO-) have been prepared by deprotonation of their respective hydroperoxides in a stream of helium buffer gas. Photodetachment with 364 nm (3.408 eV) radiation was used to measure the adiabatic electron affinities:  EA[CH_3OO, X̃^2A‘‘] = 1.161 ± 0.005 eV, EA[CD_3OO, X̃^2A‘‘] = 1.154 ± 0.004 eV, and EA[CH_3CH_2OO, X̃^2A‘‘] = 1.186 ± 0.004 eV. The photoelectron spectra yield values for the term energies:  ΔE(X̃^2A‘‘−Ã^2A‘)[CH_3OO] = 0.914 ± 0.005 eV, ΔE(X̃^2A‘‘−Ã^2A‘)[CD_3OO] = 0.913 ± 0.004 eV, and ΔE(X̃^2A‘‘−Ã^2A‘)[CH_3CH_2OO] = 0.938 ± 0.004 eV. A localized RO−O stretching mode was observed near 1100 cm^(-1) for the ground state of all three radicals, and low-frequency R−O−O bending modes are also reported. Proton-transfer kinetics of the hydroperoxides have been measured in a tandem flowing afterglow−selected ion flow tube (FA-SIFT) to determine the gas-phase acidity of the parent hydroperoxides: Δ_(acid)G_(298)(CH_3OOH) = 367.6 ± 0.7 kcal mol^(-1), Δ_(acid)G_(298)(CD_3OOH) = 367.9 ± 0.9 kcal mol^(-1), and Δ_(acid)G_(298)(CH_3CH_2OOH) = 363.9 ± 2.0 kcal mol^(-1). From these acidities we have derived the enthalpies of deprotonation: Δ_(acid)H_(298)(CH_3OOH) = 374.6 ± 1.0 kcal mol^(-1), Δ_(acid)H_(298)(CD_3OOH) = 374.9 ± 1.1 kcal mol^(-1), and Δ_(acid)H_(298)(CH_3CH_2OOH) = 371.0 ± 2.2 kcal mol^(-1). Use of the negative-ion acidity/EA cycle provides the ROO−H bond enthalpies: DH_(298)(CH_3OO−H) = 87.8 ± 1.0 kcal mol^(-1), DH_(298)(CD_3OO−H) = 87.9 ± 1.1 kcal mol^(-1), and DH_(298)(CH_3CH_2OO−H) = 84.8 ± 2.2 kcal mol^(-1). We review the thermochemistry of the peroxyl radicals, CH_3OO and CH_3CH_2OO. Using experimental bond enthalpies, DH_(298)(ROO−H), and CBS/APNO ab initio electronic structure calculations for the energies of the corresponding hydroperoxides, we derive the heats of formation of the peroxyl radicals. The “electron affinity/acidity/CBS” cycle yields Δ_fH_(298)[CH_3OO] = 4.8 ± 1.2 kcal mol^(-1) and Δ_fH_(298)[CH_3CH_2OO] = −6.8 ± 2.3 kcal mol^(-1)