No evidence on infectious DNA-based agents in pediatric acute lymphoblastic leukemia using whole metagenome shotgun sequencing.

The etiology of pediatric acute lymphatic leukemia (ALL) is still unclear. Whole-metagenome shotgun sequencing of bone marrow samples in patients with treatment-naïve ALL (n=6) was performed for untargeted investigation of bacterial and viral DNA. The control group consisted of healthy children (n=4) and children with non-oncologic diseases (n=2) undergoing bone marrow sampling. Peripheral blood of all participants was investigated at the same time. After bioinformatical elimination of potential contaminants by comparison with the employed controls, no significant amounts of microbial or viral DNA were identified.

Vascular rehabilitation in children with chronic intestinal failure reduces the risk of central-line associated bloodstream infections and catheter replacements.

BACKGROUND & AIMS: Children with chronic intestinal failure (IF) require a long-term central venous catheter (CVC) for provision of parenteral nutrition. Vascular, mechanical and infectious complications such as central line-associated bloodstream infections (CLABSI) may lead to progressive loss of venous access sites. Handling and management of CVCs therefore play an important role. Our vascular rehabilitation concept (VRC) is a core component of our intestinal rehabilitation program (IRP) and consists of an education program, optimization of skin care, catheter fixation and lock solution, and the use of hybrid technique for catheter placement. Aim of this study is to analyse the effectiveness of our VRC on CLABSI rates and need for CVC replacements. METHODS: Retrospective analysis of all children treated in our IRP that were followed up between 2018 and 2023. RESULTS: A total of 117 children with chronic IF could be included for analysis of 248864 catheter days (CD). 91 patients were referred from other hospitals (127117 CD before and 89359 CD after entry into our IRP). Children receiving primary care at our IRP (32388 CD) showed a significantly lower CLABSI and line replacement rate than patients referred from external centers (p < 0.001). After entering our IRP, CLABSI rates and need for CVC replacements per 1000 CD decreased significantly in referred patients: 1.19 to 0.26 and 1.77 to 0.59, respectively (p < 0.001). CONCLUSION: Management of paediatric chronic IF patients by an IRP with a vascular rehabilitation concept significantly lowers the rate of CLABSI episodes and the need for catheter replacements.

The capsular polysaccharide obstructs wall teichoic acid functions in Staphylococcus aureus.

BACKGROUND: The cell envelope of Staphylococcus aureus contains two major secondary cell wall glycopolymers: capsular polysaccharide (CP) and wall teichoic acid (WTA). Both the CP and the WTA are attached to the cell wall and play distinct roles in S. aureus colonization, pathogenesis, and bacterial evasion of host immune defenses. OBJECTIVE: We aimed to investigate whether CP interferes with WTA-mediated properties. METHODS: Strains with natural heterogeneous expression of CP, strains with homogeneous high CP expression and CP-deficient strains were compared to WTA deficient controls regarding WTA dependent phage binding, cell adhesion, IgG deposition, and virulence in vivo. RESULTS: WTA-mediated phage adsorption, specific antibody deposition and cell adhesion were negatively correlated with CP expression. WTA, but not CP, enhanced the bacterial burden in a mouse abscess model, while CP overexpression resulted in intermediate virulence in vivo. CONCLUSIONS: CP protects the bacteria from WTA-dependent opsonization and phage binding. This protection comes at the cost of diminished adhesion to host cells. The highly complex regulation and mostly heterogeneous expression of CP has probably evolved to ensure the survival and optimal physiological adaptation of the bacterial population as a whole.

Wall teichoic acid substitution with glucose governs phage susceptibility of Staphylococcus epidermidis.

The species- and clone-specific susceptibility of Staphylococcus cells for bacteriophages is governed by the structures and glycosylation patterns of wall teichoic acid (WTA) glycopolymers. The glycosylation-dependent phage-WTA interactions in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) have remained unknown. We report a new S. epidermidis WTA glycosyltransferase TagE whose deletion confers resistance to siphoviruses such as ΦE72 but enables binding of otherwise unbound podoviruses. S. epidermidis glycerolphosphate WTA was found to be modified with glucose in a tagE-dependent manner. TagE is encoded together with the enzymes PgcA and GtaB providing uridine diphosphate-activated glucose. ΦE72 transduced several other CoNS species encoding TagE homologs, suggesting that WTA glycosylation via TagE is a frequent trait among CoNS that permits interspecies horizontal gene transfer. Our study unravels a crucial mechanism of phage-Staphylococcus interaction and horizontal gene transfer, and it will help in the design of anti-staphylococcal phage therapies.IMPORTANCEPhages are highly specific for certain bacterial hosts, and some can transduce DNA even across species boundaries. How phages recognize cognate host cells remains incompletely understood. Phages infecting members of the genus Staphylococcus bind to wall teichoic acid (WTA) glycopolymers with highly variable structures and glycosylation patterns. How WTA is glycosylated in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) species has remained unknown. We describe that S. epidermidis glycosylates its WTA backbone with glucose, and we identify a cluster of three genes responsible for glucose activation and transfer to WTA. Their inactivation strongly alters phage susceptibility patterns, yielding resistance to siphoviruses but susceptibility to podoviruses. Many different CoNS species with related glycosylation genes can exchange DNA via siphovirus ΦE72, suggesting that glucose-modified WTA is crucial for interspecies horizontal gene transfer. Our finding will help to develop antibacterial phage therapies and unravel routes of genetic exchange.

Impact of COVID-19 on paediatric chronic intestinal failure: A tertiary care children's hospital experience.

OBJECTIVES: Paediatric patients with intestinal failure (IF) are at risk for both gastrointestinal (GI) and systemic complications, thus depending on a functioning network of multidisciplinary care. Data on the clinical impact of coronavirus disease 2019 (COVID-19) or the pandemic-related restrictions are limited. We aimed to analyse the clinical course of COVID-19 in children with IF, and to evaluate the perceived impact of the COVID-19 pandemic on IF patients and their caregivers by analysing quality of life (QoL), health-related QoL (HRQoL) and health care. METHODS: Children with IF presenting at our intestinal rehabilitation centre were enrolled and interviewed about test-proven COVID-19 infection. A standardised questionnaire was offered to all caregivers of IF patients and to two control groups (children with inflammatory bowel disease and gastrointestinal healthy children). RESULTS: Between December 2020 and November 2022, 25 out of 127 patients with IF contracted COVID-19. Forty-eight per cent had GI symptoms, 32% required additional intravenous fluids and 20% were hospitalized. Only 25% of vaccinated children showed signs of GI dysfunction, compared to 52% of unvaccinated children. Analysis of 93 questionnaires showed a negative impact on QoL and HRQoL (>66.7% and >27.8%, respectively). IF patients frequently experienced restrictions in health care, including appointments, services and supply of parenteral nutrition or medications. Caregiver burden increased significantly more often in caregivers of children with IF (p = 0.007). CONCLUSIONS: Paediatric patients with IF contracting COVID-19 have an increased risk for GI dysfunction which may be alleviated by vaccination. Children and their caregivers were highly burdened by pandemic-related restrictions and reductions in health care provision.

Quality of life and healthcare utilization during the COVID-19 pandemic are more restricted in chronically ill than in healthy children: a tertiary care children's hospital experience.

The global COVID-19 pandemic forced changes in everyday life of children and adolescents due to government containment measures, an altered healthcare accessibility and utilization, and public concern about SARS-CoV-2 transmission. Data on the challenges and impact on children and their families with chronic diseases are limited. The primary objectives of this study were to assess (i) concerns for SARS-CoV-2 infection, (ii) perceived effects on health-related and overall quality of life (HRQoL and QoL), and (iii) accessibility and utilization of healthcare, comparing families with chronically ill children to families with healthy children during the second SARS-CoV-2 infection wave in Germany. A caregiver questionnaire was designed and participation offered in the emergency department and outpatient clinic of a German tertiary care children's hospital. 45.9% of the 205 participants were majorly concerned about their children contracting a SARS-CoV-2 infection. Caregivers of chronically ill children (128/205, 62.4%) stated significantly more often a negative impact on their child's QoL (w = 0.17; p = 0.014), while caregivers of chronically ill adolescents over the age of 13 expressed significantly more frequent a negative impact on their child's HRQoL (w = 0.21; p = 0.016). Outpatient appointments for chronically ill children were significantly more often canceled (w = 0.17; p = 0.025). Caregivers of chronically ill children were significantly more likely to report that they would actively delay hospital visits for emerging health issues due to the pandemic (w = 0.12; p = 0.049).     Conclusion: Our findings underscore the importance of identifying families with chronically ill children as a vulnerable patient group with higher burdens during the COVID-19 pandemic and potential future pandemics. Healthcare providers may mitigate such burdens by ensuring reliable appointment allocation, offering contactless healthcare options, and providing tailored advice regarding vulnerabilities and preventive measures specific to their chronically ill children. What is Known: • The SARS-CoV-2 pandemic has led to significant restrictions in everyday life and both accessibility and utilization of healthcare for children and adolescents. • Chronically ill children faced exceptional challenges as they depend on regular and functioning medical care, but data comparing the pandemic's impact between chronically ill and healthy children are lacking. What is New: • The perceived impact of the SARS-CoV-2 pandemic on quality of life is more negative for chronically ill children and their health-related quality of life is more often affected compared to healthy children. • Caregivers of chronically ill children would more often delay a visit to their child's doctor during the SARS-CoV-2 pandemic and their medical appointments are more often postponed which both could increase health burdens for such vulnerable patients.

Wall Teichoic Acid Mediates Staphylococcus aureus Binding to Endothelial Cells via the Scavenger Receptor LOX-1.

The success of Staphylococcus aureus as a major cause for endovascular infections depends on effective interactions with blood-vessel walls. We have previously shown that S. aureus uses its wall teichoic acid (WTA), a surface glycopolymer, to attach to endothelial cells. However, the endothelial WTA receptor remained unknown. We show here that the endothelial oxidized low-density lipoprotein receptor 1 (LOX-1) interacts with S. aureus WTA and permits effective binding of S. aureus to human endothelial cells. Purified LOX-1 bound to isolated S. aureus WTA. Ectopic LOX-1 expression led to increased binding of S. aureus wild type but not of a WTA-deficient mutant to a cell line, and LOX-1 blockage prevented S. aureus binding to endothelial cells. Moreover, WTA and LOX-1 expression levels correlated with the efficacy of the S. aureus-endothelial interaction. Thus, LOX-1 is an endothelial ligand for S. aureus, whose blockage may help to prevent or treat severe endovascular infections.

Locally Advanced Adrenocortical Carcinoma in Children and Adolescents-Enigmatic and Challenging Cases.

BACKGROUND: Locally advanced tumors account for approximately 50% of children and adolescents with adrenocortical carcinoma (ACC), and of these, up to 50% relapse. We explored the five-item microscopic score and the pS-GRAS score for guiding management. METHODS: Data from children and adolescents with COG stage II and III ACC registered in the MET studies were included. The five-item and pS-GRAS score were retrospectively calculated. RESULTS: By December 2021, 55 patients with stage II and III (stage II n = 18, stage III n = 37) had been reported. Median age was 4.3 years [0.1-17.8], median duration of follow-up 6.0 years [0-16.7]. 3-year event-free survival (EFS) rate was 76.5% and 49.8% (p = 0.088), respectively. In stage II tumors, neither the five-item score (p = 0.872) nor pS-GRAS grouping (p = 0.218) had any effect as prognostic factors. In stage III patients, EFS was impaired in tumors with unfavorable histology according to the five-item score (100% vs. 30.8%, p = 0.018). No difference was observed for pS-GRAS groups (p = 0.798). CONCLUSIONS: In patients with COG stage III, but not stage II, the five-item score affected EFS. Further studies are needed to identify patients at risk in COG stage II.

Infection control of COVID-19 in pediatric tertiary care hospitals: challenges and implications for future pandemics.

BACKGROUND: More than 2 years into the COVID-19 pandemic, SARS-CoV-2 still impacts children's health and the management of pediatric hospitals. However, it is unclear which hygiene and infection control measures are effective and useful for pediatric hospitals. Here, we report infection control measures implemented at a tertiary care children's hospital. We evaluated frequency of SARS-CoV-2 detection in admitted patients, in-hospital transmission and infection related findings. Furthermore, we aimed to capture perspectives of health-care workers and caregivers on effectiveness and burden of infection control measures. Knowledge gained can inform management of the ongoing and future pandemics. METHODS: We designed a retrospective observational study and survey at a pediatric tertiary care referral center. Local infection control measures and respective guidelines regarding COVID-19 were reviewed. Three thousand seven hundred sixteen children under 18 years were tested for SARS-CoV-2 at the University Children's Hospital Tuebingen and data on SARS-CoV-2 transmission were retrieved from internal records. Two surveys were conducted among 219 staff members and 229 caregivers. RESULTS: Local infection control measures comprised the formation of a task force, triage, protective hygiene measures and an adaptable SARS-CoV-2 test strategy. Between January 2020 and March 2021, SARS-CoV-2 infection was detected in 37 children presenting to our hospital, 21 of these were admitted. One hospital-acquired infection occurred. About 90% of health-care staff perceived the majority of measures as effective and appropriate. However, visitor restrictions and cancellation of scheduled treatments were perceived least effective by hospital staff and as a particular burden for patients and their caregivers. Visits at the pediatric emergency department significantly decreased during the pandemic. We drafted a pandemic action plan by ranking infection control measures according to local transmission stages. CONCLUSIONS: SARS-CoV-2 infection control measures implemented in our tertiary care children's hospital were evaluated by health-care workers as mostly effective and appropriate. In particular, good communication, transparency of decision-making as well as universal masking and infection screening were assessed as successful measures of infection control management. Visitor restrictions and cancellation of routine appointments, in contrast, were perceived as a particular burden on patient care and should be avoided. An established pandemic action plan may guide children's hospitals in the future.

Sensitizing Staphylococcus aureus to antibacterial agents by decoding and blocking the lipid flippase MprF.

The pandemic of antibiotic resistance represents a major human health threat demanding new antimicrobial strategies. Multiple peptide resistance factor (MprF) is the synthase and flippase of the phospholipid lysyl-phosphatidylglycerol that increases virulence and resistance of methicillin-resistant Staphylococcus aureus (MRSA) and other pathogens to cationic host defense peptides and antibiotics. With the aim to design MprF inhibitors that could sensitize MRSA to antimicrobial agents and support the clearance of staphylococcal infections with minimal selection pressure, we developed MprF-targeting monoclonal antibodies, which bound and blocked the MprF flippase subunit. Antibody M-C7.1 targeted a specific loop in the flippase domain that proved to be exposed at both sides of the bacterial membrane, thereby enhancing the mechanistic understanding of bacterial lipid translocation. M-C7.1 rendered MRSA susceptible to host antimicrobial peptides and antibiotics such as daptomycin, and it impaired MRSA survival in human phagocytes. Thus, MprF inhibitors are recommended for new antivirulence approaches against MRSA and other bacterial pathogens.

Staphylococcus epidermidis clones express Staphylococcus aureus-type wall teichoic acid to shift from a commensal to pathogen lifestyle.

Most clonal lineages of Staphylococcus epidermidis are commensals present on human skin and in the nose. However, some globally spreading healthcare-associated and methicillin-resistant S. epidermidis (HA-MRSE) clones are major causes of difficult-to-treat implant or bloodstream infections. The molecular determinants that alter the lifestyle of S. epidermidis have remained elusive, and their identification might provide therapeutic targets. We reasoned that changes in surface-exposed wall teichoic acid (WTA) polymers of S. epidermidis, which potentially shape host interactions, may be linked to differences between colonization and infection abilities of different clones. We used a combined epidemiological and functional approach to show that while commensal clones express poly-glycerolphosphate WTA, S. epidermidis multilocus sequence type 23, which emerged in the past 15 years and is one of the main infection-causing HA-MRSE clones, contains an accessory genetic element, tarIJLM, that leads to the production of a second, Staphylococcus aureus-type WTA (poly-ribitolphosphate (RboP)). Production of RboP-WTA by S. epidermidis impaired in vivo colonization but augmented endothelial attachment and host mortality in a mouse sepsis model. tarIJLM was absent from commensal human sequence types but was found in several other HA-MRSE clones. Moreover, RboP-WTA enabled S. epidermidis to exchange DNA with S. aureus via siphovirus bacteriophages, thereby creating a possible route for the inter-species exchange of methicillin resistance, virulence and colonization factors. We conclude that tarIJLM alters the lifestyle of S. epidermidis from commensal to pathogenic and propose that RboP-WTA might be a robust target for preventive and therapeutic interventions against MRSE infections.

Odevixibat and partial external biliary diversion showed equal improvement of cholestasis in a patient with progressive familial intrahepatic cholestasis.

Untreated progressive familial intrahepatic cholestasis (PFIC) type 2, or bile salt exporter protein deficiency, frequently leads to severe pruritus, impaired growth and progressive liver fibrosis with risk of organ failure. We describe a 15-month-old male patient with severe pruritus diagnosed with PFIC type 2 enrolled in an open-label phase 2 study who received 4 weeks of treatment with odevixibat, an ileal bile acid transporter inhibitor under development for cholestatic liver disease treatment. The patient experienced reductions in serum bile acids and improvement in itching and sleep scores, and odevixibat was well tolerated. After the odevixibat study, symptoms returned and the patient underwent partial external biliary diversion (PEBD). Odevixibat treatment and PEBD produced similar normalisation of serum bile acid levels and improvements in pruritus and sleep disruptions. Thus, odevixibat appeared to be as effective as invasive PEBD in treating serum bile acids and cholestatic pruritus in this patient.

Gain-of-Function Mutations in the Phospholipid Flippase MprF Confer Specific Daptomycin Resistance.

Daptomycin, a calcium-dependent lipopeptide antibiotic whose full mode of action is still not entirely understood, has become a standard-of-care agent for treating methicillin-resistant Staphylococcus aureus (MRSA) infections. Daptomycin-resistant (DAP-R) S. aureus mutants emerge during therapy, featuring isolates which in most cases possess point mutations in the mprF gene. MprF is a bifunctional bacterial resistance protein that synthesizes the positively charged lipid lysyl-phosphatidylglycerol (LysPG) and translocates it subsequently from the inner membrane leaflet to the outer membrane leaflet. This process leads to increased positive S. aureus surface charge and reduces susceptibility to cationic antimicrobial peptides and cationic antibiotics. We characterized the most commonly reported MprF mutations in DAP-R S. aureus strains in a defined genetic background and found that only certain mutations, including the frequently reported T345A single nucleotide polymorphism (SNP), can reproducibly cause daptomycin resistance. Surprisingly, T345A did not alter LysPG synthesis, LysPG translocation, or the S. aureus cell surface charge. MprF-mediated DAP-R relied on a functional flippase domain and was restricted to daptomycin and a related cyclic lipopeptide antibiotic, friulimicin B, suggesting that the mutations modulate specific interactions with these two antibiotics. Notably, the T345A mutation led to weakened intramolecular domain interactions of MprF, suggesting that daptomycin and friulimicin resistance-conferring mutations may alter the substrate range of the MprF flippase to directly translocate these lipopeptide antibiotics or other membrane components with crucial roles in the activity of these antimicrobials. Our study points to a new mechanism used by S. aureus to resist calcium-dependent lipopeptide antibiotics and increases our understanding of the bacterial phospholipid flippase MprF.IMPORTANCE Ever since daptomycin was introduced to the clinic, daptomycin-resistant isolates have been reported. In most cases, the resistant isolates harbor point mutations in MprF, which produces and flips the positively charged phospholipid LysPG. This has led to the assumption that the resistance mechanism relies on the overproduction of LysPG, given that increased LysPG production may lead to increased electrostatic repulsion of positively charged antimicrobial compounds, including daptomycin. Here we show that the resistance mechanism is highly specific and relies on a different process that involves a functional MprF flippase, suggesting that the resistance-conferring mutations may enable the flippase to accommodate daptomycin or an unknown component that is crucial for its activity. Our report provides a new perspective on the mechanism of resistance to a major antibiotic.

Inhibition of the ATP Synthase Eliminates the Intrinsic Resistance of Staphylococcus aureus towards Polymyxins.

Staphylococcus aureus is intrinsically resistant to polymyxins (polymyxin B and colistin), an important class of cationic antimicrobial peptides used in treatment of Gram-negative bacterial infections. To understand the mechanisms underlying intrinsic polymyxin resistance in S. aureus, we screened the Nebraska Transposon Mutant Library established in S. aureus strain JE2 for increased susceptibility to polymyxin B. Nineteen mutants displayed at least 2-fold reductions in MIC, while the greatest reductions (8-fold) were observed for mutants with inactivation of either graS, graR, vraF, or vraG or the subunits of the ATP synthase (atpA, atpB, atpG, or atpH), which during respiration is the main source of energy. Inactivation of atpA also conferred hypersusceptibility to colistin and the aminoglycoside gentamicin, whereas susceptibilities to nisin, gallidermin, bacitracin, vancomycin, ciprofloxacin, linezolid, daptomycin, and oxacillin were unchanged. ATP synthase activity is known to be inhibited by oligomycin A, and the presence of this compound increased polymyxin B-mediated killing of S. aureus Our results demonstrate that the ATP synthase contributes to intrinsic resistance of S. aureus towards polymyxins and that inhibition of the ATP synthase sensitizes S. aureus to this group of compounds. These findings show that by modulation of bacterial metabolism, new classes of antibiotics may show efficacy against pathogens towards which they were previously considered inapplicable. In light of the need for new treatment options for infections with serious pathogens like S. aureus, this approach may pave the way for novel applications of existing antibiotics.IMPORTANCE Bacterial pathogens that cause disease in humans remain a serious threat to public health, and antibiotics are still our primary weapon in treating bacterial diseases. The ability to eradicate bacterial infections is critically challenged by development of resistance to all clinically available antibiotics. Polymyxins constitute an important class of antibiotics for treatment of infections caused by Gram-negative pathogens, whereas Gram-positive bacteria remain largely insusceptible towards class of antibiotics. Here we performed a whole-genome screen among nonessential genes for polymyxin intrinsic resistance determinants in Staphylococcus aureus We found that the ATP synthase is important for polymyxin susceptibility and that inhibition of the ATP synthase sensitizes S. aureus towards polymyxins. Our study provides novel insights into the mechanisms that limit polymyxin activity against S. aureus and provides valuable targets for inhibitors to potentially enable the use of polymyxins against S. aureus and other Gram-positive pathogens.

Bacterial aminoacyl phospholipids - Biosynthesis and role in basic cellular processes and pathogenicity.

The bacterial cell membrane accomplishes the controlled exchange of molecules with the extracellular space and mediates specific interactions with the environment. However, the cytoplasmic membrane also includes vulnerable targets for antimicrobial agents. A common feature of cationic antimicrobial peptides (CAMPs) produced by other bacteria or by the host immune system is to utilize the negative charge of bacterial phospholipids such as phosphatidylglycerol (PG) or cardiolipin (CL) for initial adherence and subsequent penetration into the membrane bilayer. To resist cationic antimicrobials many bacteria integrate positive charges into the membrane surface. This is accomplished by aminoacylation of negatively charged (PG) or (CL) with alanine, arginine, or lysine residues. The Multiple Peptide Resistance Factor (MprF) of Staphylococcus aureus is the prototype of a highly conserved protein family of aminoacyl phosphatidylglycerol synthases (aaPGSs) which modify PG or CL with amino acids. MprF is an oligomerizing membrane protein responsible for both, synthesis of lysyl phosphatidylglycerol (LysPG) in the inner leaflet of the cytoplasmic membrane and translocation of LysPG to the outer leaflet. This review focuses on occurrence, synthesis and function of bacterial aminoacyl phospholipids (aaPLs) and on the role of such lipids in basic cellular processes and pathogenicity. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.

Human commensals producing a novel antibiotic impair pathogen colonization.

The vast majority of systemic bacterial infections are caused by facultative, often antibiotic-resistant, pathogens colonizing human body surfaces. Nasal carriage of Staphylococcus aureus predisposes to invasive infection, but the mechanisms that permit or interfere with pathogen colonization are largely unknown. Whereas soil microbes are known to compete by production of antibiotics, such processes have rarely been reported for human microbiota. We show that nasal Staphylococcus lugdunensis strains produce lugdunin, a novel thiazolidine-containing cyclic peptide antibiotic that prohibits colonization by S. aureus, and a rare example of a non-ribosomally synthesized bioactive compound from human-associated bacteria. Lugdunin is bactericidal against major pathogens, effective in animal models, and not prone to causing development of resistance in S. aureus. Notably, human nasal colonization by S. lugdunensis was associated with a significantly reduced S. aureus carriage rate, suggesting that lugdunin or lugdunin-producing commensal bacteria could be valuable for preventing staphylococcal infections. Moreover, human microbiota should be considered as a source for new antibiotics.

The lipid-modifying multiple peptide resistance factor is an oligomer consisting of distinct interacting synthase and flippase subunits.

UNLABELLED: Phospholipids are synthesized at the inner leaflet of the bacterial cytoplasmic membrane but have to be translocated to the outer leaflet to maintain membrane lipid bilayer composition and structure. Even though phospholipid flippases have been proposed to exist in bacteria, only one such protein, MprF, has been described. MprF is a large integral membrane protein found in several prokaryotic phyla, whose C terminus modifies phosphatidylglycerol (PG), the most common bacterial phospholipid, with lysine or alanine to modulate the membrane surface charge and, as a consequence, confer resistance to cationic antimicrobial agents such as daptomycin. In addition, MprF is a flippase for the resulting lipids, Lys-PG or Ala-PG. Here we demonstrate that the flippase activity resides in the N-terminal 6 to 8 transmembrane segments of the Staphylococcus aureus MprF and that several conserved, charged amino acids and a proline residue are crucial for flippase function. MprF protects S. aureus against the membrane-active antibiotic daptomycin only when both domains are present, but the two parts do not need to be covalently linked and can function in trans. The Lys-PG synthase and flippase domains were each found to homo-oligomerize and also to interact with each other, which illustrates how the two functional domains may act together. Moreover, full-length MprF proteins formed oligomers, indicating that MprF functions as a dimer or larger oligomer. Together our data reveal how bacterial phospholipid flippases may function in the context of lipid biosynthetic processes. IMPORTANCE: Bacterial cytoplasmic membranes are crucial for maintaining and protecting cellular integrity. For instance, they have to cope with membrane-damaging agents such as cationic antimicrobial peptides (CAMPs) produced by competing bacteria (bacteriocins), secreted by eukaryotic host cells (defensins), or used as antimicrobial therapy (daptomycin). The MprF protein is found in many Gram-positive, Gram-negative, and even archaeal commensals or pathogens and confers resistance to CAMPs by modifying anionic phospholipids with amino acids, thereby compromising the membrane interaction of CAMPs. Here we describe how MprF does not only modify phospholipids but uses an additional, distinct domain for translocating the resulting lysinylated phospholipids to the outer leaflet of the membrane. We reveal critical details for the structure and function of MprF, the first dedicated prokaryotic phospholipid flippase, which may pave the way for targeting MprF with new antimicrobials that would not kill bacteria but sensitize them to antibiotics and innate host defense molecules.

Synthesis and function of phospholipids in Staphylococcus aureus.

Phospholipids are the major components of bacterial membranes, and changes in phospholipid composition affect important cellular processes such as metabolism, stress response, antimicrobial resistance, and virulence. The most prominent phospholipids in Staphylococcus aureus are phosphatidylglycerol, lysyl-phosphatidylglycerol, and cardiolipin, whose biosynthesis is mediated by a complex protein machinery. Phospholipid composition of the staphylococcal membrane has to be continuously adjusted to changing external conditions, which is achieved by a series of transcriptional and biochemical regulatory mechanisms. This mini-review outlines the current state of knowledge concerning synthesis, regulation, and function of the major staphylococcal phospholipids.

Alanyl-phosphatidylglycerol and lysyl-phosphatidylglycerol are translocated by the same MprF flippases and have similar capacities to protect against the antibiotic daptomycin in Staphylococcus aureus.

The lysinylation of negatively charged phosphatidylglycerol by MprF proteins reduces the affinity of cationic antimicrobial peptides (CAMPs) for bacterial cytoplasmic membranes and reduces the susceptibility of several Gram-positive bacterial pathogens to CAMPs. MprF of Staphylococcus aureus encompasses a lysyl-phosphatidylglycerol (Lys-PG) synthase and a Lys-PG flippase domain. In contrast, Clostridium perfringens encodes two MprF homologs which specifically synthesize alanyl-phosphatidylglycerol (Ala-PG) or Lys-PG, while only the Lys-PG synthase is fused to a putative flippase domain. It remains unknown whether cationic Lys-PG and zwitterionic Ala-PG differ in their capacities to be translocated by MprF flippases and if both can reduce CAMP susceptibility in Gram-positive bacteria. By expressing the MprF proteins of C. perfringens in an S. aureus mprF deletion mutant, we found that both lipids can be efficiently produced in S. aureus. Simultaneous expression of the Lys-PG and Ala-PG synthases led to the production of both lipids and slightly increased the overall amounts of aminoacyl phospholipids. Ala-PG production by the corresponding C. perfringens enzyme did not affect susceptibility to CAMPs such as nisin and gallidermin or to the CAMP-like antibiotic daptomycin. However, coexpression of the Ala-PG synthase with flippase domains of Lys-PG synthesizing MprF proteins led to a wild-type level of daptomycin susceptibility, indicating that Ala-PG can also protect bacterial membranes against daptomycin and suggesting that Lys-PG flippases can also translocate the related lipid Ala-PG. Thus, bacterial aminoacyl phospholipid flippases exhibit more relaxed substrate specificity and Ala-PG and Lys-PG are more similar in their capacities to modulate membrane functions than anticipated.