The carboxy terminus causes interfacial assembly of oleate hydratase on a membrane bilayer.

The soluble flavoprotein oleate hydratase (OhyA) hydrates the 9-cis double bond of unsaturated fatty acids. OhyA substrates are embedded in membrane bilayers; OhyA must remove the fatty acid from the bilayer and enclose it in the active site. Here, we show that the positively charged helix-turn-helix motif in the carboxy terminus (CTD) is responsible for interacting with the negatively charged phosphatidylglycerol (PG) bilayer. Super-resolution microscopy of Staphylococcus aureus cells expressing green fluorescent protein fused to OhyA or the CTD sequence shows subcellular localization along the cellular boundary, indicating OhyA is membrane-associated and the CTD sequence is sufficient for membrane recruitment. Using cryo-electron microscopy, we solved the OhyA dimer structure and conducted 3D variability analysis of the reconstructions to assess CTD flexibility. Our surface plasmon resonance experiments corroborated that OhyA binds the PG bilayer with nanomolar affinity and we found the CTD sequence has intrinsic PG binding properties. We determined that the nuclear magnetic resonance structure of a peptide containing the CTD sequence resembles the OhyA crystal structure. We observed intermolecular NOE from PG liposome protons next to the phosphate group to the CTD peptide. The addition of paramagnetic MnCl2 indicated the CTD peptide binds the PG surface but does not insert into the bilayer. Molecular dynamics simulations, supported by site-directed mutagenesis experiments, identify key residues in the helix-turn-helix that drive membrane association. The data show that the OhyA CTD binds the phosphate layer of the PG surface to obtain bilayer-embedded unsaturated fatty acids.

S. aureus drives itch and scratch-induced skin damage through a V8 protease-PAR1 axis.

Itch is an unpleasant sensation that evokes a desire to scratch. The skin barrier is constantly exposed to microbes and their products. However, the role of microbes in itch generation is unknown. Here, we show that Staphylococcus aureus, a bacterial pathogen associated with itchy skin diseases, directly activates pruriceptor sensory neurons to drive itch. Epicutaneous S. aureus exposure causes robust itch and scratch-induced damage. By testing multiple isogenic bacterial mutants for virulence factors, we identify the S. aureus serine protease V8 as a critical mediator in evoking spontaneous itch and alloknesis. V8 cleaves proteinase-activated receptor 1 (PAR1) on mouse and human sensory neurons. Targeting PAR1 through genetic deficiency, small interfering RNA (siRNA) knockdown, or pharmacological blockade decreases itch and skin damage caused by V8 and S. aureus exposure. Thus, we identify a mechanism of action for a pruritogenic bacterial factor and demonstrate the potential of inhibiting V8-PAR1 signaling to treat itch.

DNA rehybridization drives product release from Cas9 ribonucleoprotein to enable multiple-turnover cleavage.

The RNA-guided Cas9 endonuclease from Staphylococcus aureus (SauCas9) can catalyze multiple-turnover reactions whereas Cas9 from Streptococcus pyogenes (SpyCas9) is a single-turnover enzyme. Here we dissect the mechanism of multiple-turnover catalysis by SauCas9 and elucidate its molecular basis. We show that the multiple-turnover catalysis does not require more than stoichiometric RNA guides to Cas9 nuclease. Rather, the RNA-guide loaded ribonucleoprotein (RNP) is the reactive unity that is slowly released from product and recycled in the subsequent reaction. The mechanism that RNP is recycled for multiple-turnover reaction entails the unwinding of the RNA:DNA duplex in the R-loop. We argue that DNA rehybridization is required for RNP release by supplementing the energy cost in the process. Indeed, turnover is arrested when DNA rehybridization is suppressed. Further, under higher salt conditions, both SauCas9 and SpyCas9 showed increased turnover, and engineered SpyCas9 nucleases that form fewer direct or hydrogen bonding interactions with target DNA became multiple-turnover enzymes. Thus, these results indicate that for both SpyCas9 and SauCas9, turnover is determined by the energetic balance of the post-chemistry RNP-DNA interaction. Due to the conserved protein core folds, the mechanism underpinning turnover we establish here is likely operant in all Cas9 nucleases.

Regulatory mechanism of formaldehyde release in heme degradation catalyzed by Staphylococcus aureus IsdG.

IsdG-type enzymes catalyze the noncanonical degradation of heme to iron, staphylobilin (SB), and formaldehyde (HCHO), presumably by binding heme in an unusually distorted conformation. Their unique mechanism has been elucidated for MhuD from Mycobacterium tuberculosis, revealing an unusual ring opening of hydroxyheme by dioxygenation. A similar mechanism has been postulated for other IsdG enzymes; however, MhuD, which is special as an IsdG-type enzyme, retains a formyl group in the linearized tetrapyrrole. Recent reports on Staphylococcus aureus IsdG have suggested the formation of SB retaining a formyl group (formyl-SB), but its identification is preliminary. Furthermore, the reaction properties of formyl-SB and the mechanism of HCHO release remain unclear. In this study, the complex reaction of S. aureus IsdG was reexamined to elucidate its mechanism, including the identification of reaction products and their control mechanisms. Depending on the reaction conditions, IsdG produced both SB and formyl-SB as the main product, the latter of which was isolated and characterized by MS and NMR measurements. The formyl-SB product was generated upon the reaction between hydroxyheme-IsdG and O2 without reduction, indicating the dioxygenation mechanism as found for MhuD. Under reducing conditions, hydroxyheme-IsdG was converted also to SB and HCHO by activating another O2 molecule. These results provide the first overview of the complicated IsdG reaction. The heme distortion in the IsdG-type enzymes is shown to generally promote ring cleavage by dioxygenation. The presence or absence of HCHO release can be influenced by many factors, and the direct identification of S. aureus heme catabolites is of interest.

MerA functions as a hypothiocyanous acid reductase and defense mechanism in Staphylococcus aureus.

The major pathogen Staphylococcus aureus has to cope with host-derived oxidative stress to cause infections in humans. Here, we report that S. aureus tolerates high concentrations of hypothiocyanous acid (HOSCN), a key antimicrobial oxidant produced in the respiratory tract. We discovered that the flavoprotein disulfide reductase (FDR) MerA protects S. aureus from this oxidant by functioning as a HOSCN reductase, with its deletion sensitizing bacteria to HOSCN. Crystal structures of homodimeric MerA (2.4 Å) with a Cys43 -Cys48 intramolecular disulfide, and reduced MerACys43 S (1.6 Å) showed the FAD cofactor close to the active site, supporting that MerA functions as a group I FDR. MerA is controlled by the redox-sensitive repressor HypR, which we show to be oxidized to intermolecular disulfides under HOSCN stress, resulting in its inactivation and derepression of merA transcription to promote HOSCN tolerance. Our study highlights the HOSCN tolerance of S. aureus and characterizes the structure and function of MerA as a major HOSCN defense mechanism. Crippling the capacity to respond to HOSCN may be a novel strategy for treating S. aureus infections.

The enzyme activity of sortase A is regulated by phosphorylation in Staphylococcus aureus.

In many Gram-positive bacteria, the transpeptidase enzyme sortase A (SrtA) anchors surface proteins to cell wall and plays a critical role in the bacterial pathogenesis. Here, we show that in Staphylococcus aureus, an important human pathogen, the SrtA is phosphorylated by serine/threonine protein kinase Stk1. S. aureus SrtA can also be phosphorylated by small-molecule phosphodonor acetyl phosphate (AcP) in vitro. We determined that various amino acid residues of S. aureus SrtA are subject to phosphorylation, primarily on its catalytic site residue cysteine-184 in the context of a bacterial cell lysate. Both Stk1 and AcP-mediated phosphorylation inhibited the enzyme activity of SrtA in vitro. Consequently, deletion of gene (i.e. stp1) encoding serine/threonine phosphatase Stp1, the corresponding phosphatase of Stk1, caused an increase in the phosphorylation level of SrtA. The stp1 deletion mutant mimicked the phenotypic traits of srtA deletion mutant (i.e. attenuated growth where either haemoglobin or haem as a sole iron source and reduced liver infections in a mouse model of systemic infection). Importantly, the phenotypic defects of the stp1 deletion mutant can be alleviated by overexpressing srtA. Taken together, our finding suggests that phosphorylation plays an important role in modulating the activity of SrtA in S. aureus.

Structural basis of broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Broad-spectrum ß-lactam antibiotic resistance in Staphylococcus aureus is a global healthcare burden1,2. In clinical strains, resistance is largely controlled by BlaR13, a receptor that senses ß-lactams through the acylation of its sensor domain, inducing transmembrane signalling and activation of the cytoplasmic-facing metalloprotease domain4. The metalloprotease domain has a role in BlaI derepression, inducing blaZ (ß-lactamase PC1) and mecA (ß-lactam-resistant cell-wall transpeptidase PBP2a) expression3-7. Here, overcoming hurdles in isolation, we show that BlaR1 cleaves BlaI directly, as necessary for inactivation, with no requirement for additional components as suggested previously8. Cryo-electron microscopy structures of BlaR1-the wild type and an autocleavage-deficient F284A mutant, with or without ß-lactam-reveal a domain-swapped dimer that we suggest is critical to the stabilization of the signalling loops within. BlaR1 undergoes spontaneous autocleavage in cis between Ser283 and Phe284 and we describe the catalytic mechanism and specificity underlying the self and BlaI cleavage. The structures suggest that allosteric signalling emanates from ß-lactam-induced exclusion of the prominent extracellular loop bound competitively in the sensor-domain active site, driving subsequent dynamic motions, including a shift in the sensor towards the membrane and accompanying changes in the zinc metalloprotease domain. We propose that this enhances the expulsion of autocleaved products from the active site, shifting the equilibrium to a state that is permissive of efficient BlaI cleavage. Collectively, this study provides a structure of a two-component signalling receptor that mediates action-in this case, antibiotic resistance-through the direct cleavage of a repressor.

A New 1,2,3-Triazole Scaffold with Improved Potency against Staphylococcus aureus Biotin Protein Ligase.

Staphylococcus aureus, a key ESKAPE bacteria, is responsible for most blood-based infections and, as a result, is a major economic healthcare burden requiring urgent attention. Here, we report in silico docking, synthesis, and assay of N1-diphenylmethyl triazole-based analogues (7-13) designed to interact with the entire binding site of S. aureus biotin protein ligase (SaBPL), an enzyme critical for the regulation of gluconeogenesis and fatty acid biosynthesis. The second aryl ring of these compounds enhances both SaBPL potency and whole cell activity against S. aureus relative to previously reported mono-benzyl triazoles. Analogues 12 and 13, with added substituents to better interact with the adenine binding site, are particularly potent, with Ki values of 6.01 ± 1.01 and 8.43 ± 0.73 nM, respectively. These analogues are the most active triazole-based inhibitors reported to date and, importantly, inhibit the growth of a clinical isolate strain of S. aureus ATCC 49775, with minimum inhibitory concentrations of 1 and 8 µg/mL, respectively.

Discovery of an ʟ-amino acid ligase implicated in Staphylococcal sulfur amino acid metabolism.

Enzymes involved in Staphylococcus aureus amino acid metabolism have recently gained traction as promising targets for the development of new antibiotics, however, not all aspects of this process are understood. The ATP-grasp superfamily includes enzymes that predominantly catalyze the ATP-dependent ligation of various carboxylate and amine substrates. One subset, ʟ-amino acid ligases (LALs), primarily catalyze the formation of dipeptide products in Gram-positive bacteria, however, their involvement in S. aureus amino acid metabolism has not been investigated. Here, we present the characterization of the putative ATP-grasp enzyme (SAOUHSC_02373) from S. aureus NCTC 8325 and its identification as a novel LAL. First, we interrogated the activity of SAOUHSC_02373 against a panel of ʟ-amino acid substrates. As a result, we identified SAOUHSC_02373 as an LAL with high selectivity for ʟ-aspartate and ʟ-methionine substrates, specifically forming an ʟ-aspartyl-ʟ-methionine dipeptide. Thus, we propose that SAOUHSC_02373 be assigned as ʟ-aspartate-ʟ-methionine ligase (LdmS). To further understand this unique activity, we investigated the mechanism of LdmS by X-ray crystallography, molecular modeling, and site-directed mutagenesis. Our results suggest that LdmS shares a similar mechanism to other ATP-grasp enzymes but possesses a distinctive active site architecture that confers selectivity for the ʟ-Asp and ʟ-Met substrates. Phylogenetic analysis revealed LdmS homologs are highly conserved in Staphylococcus and closely related Gram-positive Firmicutes. Subsequent genetic analysis upstream of the ldmS operon revealed several trans-acting regulatory elements associated with control of Met and Cys metabolism. Together, these findings support a role for LdmS in Staphylococcal sulfur amino acid metabolism.

Staphylococcal self-loading helicases couple the staircase mechanism with inter domain high flexibility.

Replication is a crucial cellular process. Replicative helicases unwind DNA providing the template strand to the polymerase and promoting replication fork progression. Helicases are multi-domain proteins which use an ATPase domain to couple ATP hydrolysis with translocation, however the role that the other domains might have during translocation remains elusive. Here, we studied the unexplored self-loading helicases called Reps, present in Staphylococcus aureus pathogenicity islands (SaPIs). Our cryoEM structures of the PriRep5 from SaPI5 (3.3 Å), the Rep1 from SaPI1 (3.9 Å) and Rep1-DNA complex (3.1Å) showed that in both Reps, the C-terminal domain (CTD) undergoes two distinct movements respect the ATPase domain. We experimentally demonstrate both in vitro and in vivo that SaPI-encoded Reps need key amino acids involved in the staircase mechanism of translocation. Additionally, we demonstrate that the CTD's presence is necessary for the maintenance of full ATPase and helicase activities. We speculate that this high interdomain flexibility couples Rep's activities as initiators and as helicases.

Staphylococcus aureus cell wall maintenance - the multifaceted roles of peptidoglycan hydrolases in bacterial growth, fitness, and virulence.

Staphylococcus aureus is an important human and livestock pathogen that is well-protected against environmental insults by a thick cell wall. Accordingly, the wall is a major target of present-day antimicrobial therapy. Unfortunately, S. aureus has mastered the art of antimicrobial resistance, as underscored by the global spread of methicillin-resistant S. aureus (MRSA). The major cell wall component is peptidoglycan. Importantly, the peptidoglycan network is not only vital for cell wall function, but it also represents a bacterial Achilles' heel. In particular, this network is continuously opened by no less than 18 different peptidoglycan hydrolases (PGHs) encoded by the S. aureus core genome, which facilitate bacterial growth and division. This focuses attention on the specific functions executed by these enzymes, their subcellular localization, their control at the transcriptional and post-transcriptional levels, their contributions to staphylococcal virulence and their overall importance in bacterial homeostasis. As highlighted in the present review, our understanding of the different aspects of PGH function in S. aureus has been substantially increased over recent years. This is important because it opens up new possibilities to exploit PGHs as innovative targets for next-generation antimicrobials, passive or active immunization strategies, or even to engineer them into effective antimicrobial agents.

Domain architecture and catalysis of the Staphylococcus aureus fatty acid kinase.

Fatty acid kinase (Fak) is a two-component enzyme that generates acyl-phosphate for phospholipid synthesis. Fak consists of a kinase domain protein (FakA) that phosphorylates a fatty acid enveloped by a fatty acid binding protein (FakB). The structural basis for FakB function has been established, but little is known about FakA. Here, we used limited proteolysis to define three separate FakA domains: the amino terminal FakA_N, the central FakA_L, and the carboxy terminal FakA_C. The isolated domains lack kinase activity, but activity is restored when FakA_N and FakA_L are present individually or connected as FakA_NL. The X-ray structure of the monomeric FakA_N captures the product complex with ADP and two Mg2+ ions bound at the nucleotide site. The FakA_L domain encodes the dimerization interface along with conserved catalytic residues Cys240, His282, and His284. AlphaFold analysis of FakA_L predicts the catalytic residues are spatially clustered and pointing away from the dimerization surface. Furthermore, the X-ray structure of FakA_C shows that it consists of two subdomains that are structurally related to FakB. Analytical ultracentrifugation demonstrates that FakA_C binds FakB, and site-directed mutagenesis confirms that a positively charged wedge on FakB meshes with a negatively charged groove on FakA_C. Finally, small angle X-ray scattering analysis is consistent with freely rotating FakA_N and FakA_C domains tethered by flexible linkers to FakA_L. These data reveal specific roles for the three independently folded FakA protein domains in substrate binding and catalysis.

Dihydroxy-Acid Dehydratases From Pathogenic Bacteria: Emerging Drug Targets to Combat Antibiotic Resistance.

There is an urgent global need for the development of novel therapeutics to combat the rise of various antibiotic-resistant superbugs. Enzymes of the branched-chain amino acid (BCAA) biosynthesis pathway are an attractive target for novel anti-microbial drug development. Dihydroxy-acid dehydratase (DHAD) is the third enzyme in the BCAA biosynthesis pathway. It relies on an Fe-S cluster for catalytic activity and has recently also gained attention as a catalyst in cell-free enzyme cascades. Two types of Fe-S clusters have been identified in DHADs, i.e. [2Fe-2S] and [4Fe-4S], with the latter being more prone to degradation in the presence of oxygen. Here, we characterise two DHADs from bacterial human pathogens, Staphylococcus aureus and Campylobacter jejuni (SaDHAD and CjDHAD). Purified SaDHAD and CjDHAD are virtually inactive, but activity could be reversibly reconstituted in vitro (up to ∼19,000-fold increase with kcat as high as ∼6.7 s-1 ). Inductively-coupled plasma-optical emission spectroscopy (ICP-OES) measurements are consistent with the presence of [4Fe-4S] clusters in both enzymes. N-isopropyloxalyl hydroxamate (IpOHA) and aspterric acid are both potent inhibitors for both SaDHAD (Ki =7.8 and 51.6 µM, respectively) and CjDHAD (Ki =32.9 and 35.1 µM, respectively). These compounds thus present suitable starting points for the development of novel anti-microbial chemotherapeutics.

Altered Dynamics of S. aureus Phosphofructokinase via Bond Restraints at Two Distinct Allosteric Binding Sites.

The effect of perturbation at the allosteric site was investigated through several replicas of molecular dynamics (MD) simulations conducted on bacterial phosphofructokinase (SaPFK). In our previous work, an alternative binding site was estimated to be allosteric in addition to the experimentally reported one. To highlight the effect of both allosteric sites on receptor's dynamics, MD runs were carried out on apo forms with and without perturbation. Perturbation was achieved via incorporating multiple bond restraints for residue pairs located at the allosteric site. Restraints applied to the predicted site caused one dimer to stiffen, whereas an increase in mobility was detected in the same dimer when the experimentally resolved site was restrained. Fluctuations in Cα-Cα distances which is used to disclose residues with high potential of communication indicated a marked increase in signal transmission within each dimer as the receptor switched to a restrained state. Cross-correlation of positional fluctuations indicated an overall decrease in the magnitude of both positive and negative correlations when restraints were employed on the predicted allosteric site whereas an exact opposite effect was observed for the reported site. Finally, mutual correspondence between positional fluctuations noticeably increased with restraints on predicted allosteric site, whereas an opposite effect was observed for restraints applied on experimentally reported one. In view of these findings, it is clear that the perturbation of either one of two allosteric sites effected the dynamics of the receptor with a distinct and contrasting character.

Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m1A22-tRNA methyltransferase TrmK.

The enzyme m1A22-tRNA methyltransferase (TrmK) catalyzes the transfer of a methyl group to the N1 of adenine 22 in bacterial tRNAs. TrmK is essential for Staphylococcus aureus survival during infection but has no homolog in mammals, making it a promising target for antibiotic development. Here, we characterize the structure and function of S. aureus TrmK (SaTrmK) using X-ray crystallography, binding assays, and molecular dynamics simulations. We report crystal structures for the SaTrmK apoenzyme as well as in complexes with methyl donor SAM and co-product product SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favorable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated for by a large favorable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. In addition, activity assays for SaTrmK-catalyzed methylation of A22 mutants of tRNALeu demonstrate that the adenine at position 22 is absolutely essential. In silico screening of compounds suggested the multifunctional organic toxin plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS data indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92 in the vicinity of the SAM-binding site as the sole residue modified. These results identify a cryptic binding pocket of SaTrmK, laying a foundation for future structure-based drug discovery.

Catabolic Ornithine Carbamoyltransferase Activity Facilitates Growth of Staphylococcus aureus in Defined Medium Lacking Glucose and Arginine.

Previous studies have found that arginine biosynthesis in Staphylococcus aureus is repressed via carbon catabolite repression (CcpA), and proline is used as a precursor. Unexpectedly, however, robust growth of S. aureus is not observed in complete defined medium lacking both glucose and arginine (CDM-R). Mutants able to grow on agar-containing defined medium lacking arginine (CDM-R) were selected and found to contain mutations within ahrC, encoding the canonical arginine biosynthesis pathway repressor (AhrC), or single nucleotide polymorphisms (SNPs) upstream of the native arginine deiminase (ADI) operon arcA1B1D1C1. Reverse transcription-PCR (RT-PCR) studies found that mutations within ccpA or ahrC or SNPs identified upstream of arcA1B1D1C1 increased the transcription of both arcB1 and argGH, encoding ornithine carbamoyltransferase and argininosuccinate synthase/lyase, respectively, facilitating arginine biosynthesis. Furthermore, mutations within the AhrC homologue argR2 facilitated robust growth within CDM-R. Complementation with arcB1 or arcA1B1D1C1, but not argGH, rescued growth in CDM-R. Finally, supplementation of CDM-R with ornithine stimulated growth, as did mutations in genes (proC and rocA) that presumably increased the pyrroline-5-carboxylate and ornithine pools. Collectively, these data suggest that the transcriptional regulation of ornithine carbamoyltransferase and, in addition, the availability of intracellular ornithine pools regulate arginine biosynthesis in S. aureus in the absence of glucose. Surprisingly, ~50% of clinical S. aureus isolates were able to grow in CDM-R. These data suggest that S. aureus is selected to repress arginine biosynthesis in environments with or without glucose; however, mutants may be readily selected that facilitate arginine biosynthesis and growth in specific environments lacking arginine. IMPORTANCE Staphylococcus aureus can cause infection in virtually any niche of the human host, suggesting that it has significant metabolic versatility. Indeed, bioinformatic analysis suggests that it has the biosynthetic capability to synthesize all 20 amino acids. Paradoxically, however, it is conditionally auxotrophic for several amino acids, including arginine. Studies in our laboratory are designed to assess the biological function of amino acid auxotrophy in this significant pathogen. This study reveals that the metabolic block repressing arginine biosynthesis in media lacking glucose is the transcriptional repression of ornithine carbamoyltransferase encoded by arcB1 within the native arginine deiminase operon in addition to limited intracellular pools of ornithine. Surprisingly, approximately 50% of S. aureus clinical isolates can grow in media lacking arginine, suggesting that mutations are selected in S. aureus that allow growth in particular niches of the human host.

Genomic Analysis of the Unusual Staphylococcus aureus ST630 Isolates Harboring WTA Glycosyltransferase Genes tarM and tagN.

Staphylococcus aureus (S. aureus) can cause a broad spectrum of diseases ranging from skin infections to life-threatening diseases in both community and hospital settings. The surface-exposed wall teichoic acid (WTA) has a strong impact on host interaction, pathogenicity, horizontal gene transfer, and biofilm formation in S. aureus. The unusual S. aureus ST630 strains containing both ribitol-phosphate (RboP) WTA glycosyltransferase gene tarM and glycerol-phosphate (GroP) WTA glycosyltransferase gene tagN have been found recently. Native PAGE analysis showed that the WTA of tagN, tarM-encoding ST630 strains migrated slower than that of non-tagN-encoding ST630 strains, indicating the differences in WTA structure. Some mobile genetic elements (MGEs) such as the unique GroP-WTA biosynthetic gene cluster (SaGroWI), SCCmec element, and prophages that probably originated from the CoNS were identified in tagN, tarM-encoding ST630 strains. The SaGroWI element was first defined in S. aureus ST395 strain, which was refractory to exchange MGEs with typical RboP-WTA expressing S. aureus but could undergo horizontal gene transfer events with other species and genera via the specific bacteriophage Φ187. Overall, our data indicated that this rare ST630 was prone to acquire DNA from CoNS and might serve as a novel hub for the exchange of MGEs between CoNS and S. aureus. IMPORTANCE The structure of wall-anchored glycopolymers wall teichoic acid (WTA) produced by most Gram-positive bacteria is highly variable. While most dominant Staphylococcus aureus lineages produce poly-ribitol-phosphate (RboP) WTA, the tagN, tarM-encoding ST630 lineage probably has a poly-glycerol-phosphate (GroP) WTA backbone like coagulase-negative staphylococci (CoNS). There is growing evidence that staphylococcal horizontal gene transfer depends largely on transducing helper phages via WTA as the receptor. The structural difference of WTA greatly affects the transfer of mobile genetic elements among various bacteria. With the growing advances in sequencing and analysis technologies, genetic analysis has revolutionized research activities in the field of the important pathogen S. aureus. Here, we analyzed the molecular characteristics of ST630 and found an evolutionary link between ST630 and CoNS. Elucidating the genetic information of ST630 lineage will contribute to understanding the emergence and diversification of new pathogenic strains in S. aureus.

Prior Carriage Predicts Intensive Care Unit Infections Caused by Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae.

Intensive care unit-acquired infection (ICU-AI) and extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) carriage are a major concern worldwide. Our objective was to investigate the impact of ESBL-PE carriage on ICU-AI. Our study was prospective, observational, and noninterventional. It was conducted over a 5-year period (Jan 2013-Dec 2017) in the medical-surgical intensive care unit of the Cayenne General Hospital (French Amazonia). During the study period, 1,340 patients were included, 271 (20.2%) developed ICU-AI, and 16.2% of these were caused by ESBL-PE. The main sites of ICU-AI were ventilator-associated pneumonia (35.8%) and primary bloodstream infection (29.8%). The main responsible microorganisms were Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae (ESBL-P in 35.8% of isolates), and Enterobacter cloacae (ESBL-P in 29.8% of isolates). Prior ESBL-PE carriage was diagnosed in 27.6% of patients with ICU-AI. In multivariable analysis, the sole factor associated with ESBL-PE as the responsible organism of ICU-AI was ESBL-PE carriage before ICU-AI (P < 0.001; odds ratio: 7.9 95% CI: 3.4-18.9). ESBL-PE carriers (74 patients) developed ICU-AI which was caused by ESBL-PE in 32 cases (43.2%). This proportion of patients carrying ESBL-PE who developed ICU-AI to the same microorganism was 51.2% in ESBL-P K. pneumoniae, 5.6% in ESBL-P Escherichia coli, and 40% in ESBL-P Enterobacter spp. NPV of ESBL-PE carriage to predict ICU-AI caused by ESBL-PE was above 94% and PPV was above 43%. Carriage of ESBL-P K pneumoniae and Enterobacter spp. is a strong predictor of ICU-AI caused by these two microorganisms.

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.

Zinc(II) complexes of 3,5-dibromo-salicylaldehyde and α-diimines: Synthesis, characterization and in vitro and in silico biological profile.

The synthesis of five neutral zinc(II) complexes of 3,5-dibromo-salicyladehyde (3,5-diBr-saloH) in the presence of nitrogen-donor co-ligands 2,2'-bipyridine (bipy), 1,10-phenanthroline (phen), 2,9-dimethyl-1,10-phenanthroline (neoc), or 2,2'-bipyridylamine (bipyam) was undertaken and complexes [Zn(3,5-diBr-salo)2(H2O)2] (1), [Zn(3,5-diBr-salo)2(bipy)] (2), [Zn(3,5-diBr-salo)2(phen)].3,5-diBr-saloΗ (3), [Zn(3,5-diBr-salo)2(neoc)] (4) and [Zn(3,5-diBr-salo)2(bipyam)] (5) were characterized by various techniques. The crystal structures of complexes 3 and 5 were determined by X-ray crystallography, revealing the co-existence of two different coordination modes of 3,5-diBr-salo- ligands. The new complexes show selective in vitro antibacterial activity against two Gram-positive and two Gram-negative bacterial strains. The complexes may scavenge 1,1-diphenyl-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radicals and reduce H2O2. The complexes may intercalate in-between the calf-thymus DNA-bases and have exhibited low-to-moderate ability to cleave supercoiled circular pBR322 plasmid DNA. The complexes may bind tightly and reversibly to bovine and human serum albumins. In order to explain the in vitro activity of the compounds, molecular docking studies were adopted on the crystal structure of calf-thymus DNA, human and bovine serum albumin, Escherichia coli and Staphylococcus aureus DNA-gyrase, 5-lipoxygenase, and 5-lipoxygenase activating protein. The employed in silico studies aimed to explore the ability of the compounds to bind to these target biomacromolecules, establishing a possible mechanism of action and were in accordance with the in vitro studies.