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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.

Methicillin-resistant Staphylococcus aureus (MRSA) exemplifies high-level antibiotic resistance in this major human pathogen. Its resistance to chloramphenicol is majorly conferred by enzymatic inactivation via chloramphenicol acetyltransferases (CATs). This modification sterically blocks the antibiotic’s ribosomal binding and thus neutralizes its inhibitory potency. Although CATs have been structurally studied across diverse bacteria species, the structures of S. aureus CATs (saCATs) have remained uncharacterized. To address this gap and elucidate species-specific resistance mechanisms, we determined the first high-resolution crystal structure of saCAT1, the prototypical saCAT enzyme. Structural analysis delineates the active site architecture and reveals the molecular basis for substrate recognition of both chloramphenicol and fusidic acid (FA). Further enzymatic assays demonstrated that the K m value against chloramphenicol is 16.9 µM, and the K i value of the inhibitor FA is 83.7 µM, indicating that the inhibitory capacity of FA is relatively limited. These findings provide an essential structural framework for understanding chloramphenicol resistance in S. aureus and facilitate the rational design of novel antimicrobial strategies to combat multidrug-resistant pathogens.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has rapidly spread through various routes. A genomic analysis of clinical MRSA samples revealed an unknown protein, Sav2152, predicted to be a haloacid dehalogenase (HAD)-like hydrolase, making it a potential candidate for a novel drug target. In this study, we determined the crystal structure of Sav2152, which consists of a C2-type cap domain and a core domain. The core domain contains four motifs involved in phosphatase activity that depend on the presence of Mg2+ ions. Specifically, residues D10, D12, and D233, which closely correspond to key residues in structurally homolog proteins, are responsible for binding to the metal ion and are known to play critical roles in phosphatase activity. Our findings indicate that the Mg2+ ion known to stabilize local regions surrounding it, however, paradoxically, destabilizes the local region. Through mutant screening, we identified D10 and D12 as crucial residues for metal binding and maintaining structural stability via various uncharacterized intra-protein interactions, respectively. Substituting D10 with Ala effectively prevents the interaction with Mg2+ ions. The mutation of D12 disrupts important structural associations mediated by D12, leading to a decrease in the stability of Sav2152 and an enhancement in binding affinity to Mg2+ ions. Additionally, our study revealed that D237 can replace D12 and retain phosphatase activity. In summary, our work uncovers the novel role of metal ions in HAD-like phosphatase activity.

Staphylococcus aureus is an versatile pathogen that can cause life-threatening infections. Depending on the clinical setting, up to 50% of S. aureus infections are caused by methicillin-resistant strains (MRSA) that in most cases are resistant to many other antibiotics, making treatment difficult. The emergence of community-acquired MRSA drastically changed the picture by increasing the risk of MRSA infections. Horizontal transfer of genes encoding for antibiotic resistance or virulence factors is a major concern of multidrug-resistant S. aureus infections and epidemiology. We identified and characterized a type III-like restriction system present in clinical S. aureus strains that prevents transformation with DNA from other bacterial species. Interestingly, our analysis revealed that some clinical MRSA strains are deficient in this restriction system, and thus are hypersusceptible to the horizontal transfer of DNA from other species, such as Escherichia coli, and could easily acquire a vancomycin-resistance gene from enterococci. Inactivation of this restriction system dramatically increases the transformation efficiency of clinical S. aureus strains, opening the field of molecular genetic manipulation of these strains using DNA of exogenous origin.

A method for the rapid detection of methicillin-sensitive and -resistant Staphylococcus aureus (MSSA and MRSA, respectively) and methicillin-resistant coagulase-negative staphylococci (MRCoNS) with a straightforward sample preparation protocol of blood cultures using an automated homogeneous polymerase chain reaction (PCR) assay, the GenomEra™ MRSA/SA (Abacus Diagnostica Oy, Turku, Finland), is presented. In total, 316 BacT/Alert (bioMérieux, Marcy l’Etoile, France) and 433 BACTEC (Becton Dickinson, Sparks, MD, USA) blood culture bottles were analyzed, including 725 positive cultures containing Gram-positive cocci in clusters ( n  = 419) and other Gram stain forms ( n  = 361), as well as 24 signal- and growth-negative bottles. Detection sensitivities for MSSA, MRSA, and MRCoNS were 99.4 % (158/159), 100.0 % (9/9), and 99.3 % (132/133), respectively. One false-positive MRSA result was detected from a non-staphylococci-containing bottle, yielding a specificity of 99.8 %. The lowest detectable amount of viable cells in the blood culture sample was 4 × 10 4  CFU/mL. The results were available within one hour after microbial growth detection and the two-step, time-resolved fluorometric (TRF) measurement mode employed by the GenomEra CDX™ instrument showed no interference from blood, charcoal, or culture media. The method described lacks all sample purification steps and allows reliable and simplified pathogen detection also in clinical microbiology laboratory settings without specialized molecular microbiology competence.

Broad-spectrum β-lactam antibiotic resistance in Staphylococcus aureus is a global healthcare burden 1 , 2 . In clinical strains, resistance is largely controlled by BlaR1 3 , a receptor that senses β-lactams through the acylation of its sensor domain, inducing transmembrane signalling and activation of the cytoplasmic-facing metalloprotease domain 4 . The metalloprotease domain has a role in BlaI derepression, inducing blaZ (β-lactamase PC1) and mecA (β-lactam-resistant cell-wall transpeptidase PBP2a) expression 3 – 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 previously 8 . Cryo-electron microscopy structures of BlaR1—the wild type and an autocleavage-deficient F284A mutant, with or without β-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 β-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. Cryo-electron microscopy structures of Staphylococcus aureus BlaR1 reveal dynamic signalling states regulating broad spectrum β-lactam antibiotic resistance through cleavage of the transcriptional repressor BlaI and induced expression of the β-lactamase blaZ and the β-lactam-resistant cell-wall transpeptidase mecA .

Chronic infections are difficult to treat with antibiotics but are caused primarily by drug-sensitive pathogens. Dormant persister cells that are tolerant to killing by antibiotics are responsible for this apparent paradox. Persisters are phenotypic variants of normal cells and pathways leading to dormancy are redundant, making it challenging to develop anti-persister compounds. Biofilms shield persisters from the immune system, suggesting that an antibiotic for treating a chronic infection should be able to eradicate the infection on its own. We reasoned that a compound capable of corrupting a target in dormant cells will kill persisters. The acyldepsipeptide antibiotic (ADEP4) has been shown to activate the ClpP protease, resulting in death of growing cells. Here we show that ADEP4-activated ClpP becomes a fairly nonspecific protease and kills persisters by degrading over 400 proteins, forcing cells to self-digest. Null mutants of clpP arise with high probability, but combining ADEP4 with rifampicin produced complete eradication of Staphylococcus aureus biofilms in vitro and in a mouse model of a chronic infection. Our findings indicate a general principle for killing dormant cells—activation and corruption of a target, rather than conventional inhibition. Eradication of a biofilm in an animal model by activating a protease suggests a realistic path towards developing therapies to treat chronic infections. Dormant bacterial persister cells evade antibiotic destruction and their survival gives rise to some chronic infections; this study reveals that persister cells can be eradicated with a compound activating the bacterial protease ClpP, providing an effective biofilm treatment in vitro and in mouse chronic infection models. An anti-persister antibiotic Concerns about the ability of today's antibiotics to cope with future infections are compounded by the dual nature of the bacterial response to the drugs. Some bacteria develop genetic resistance, but others become tolerant, able to survive in the presence of antibiotics by forming dormant cells known as persisters in which the enzymatic targets of the antibiotics are inactive. Kim Lewis and colleagues sought compounds with the potential to kill persisters by corrupting targets within these energy-limited cells. They demonstrate that the acyldepsipeptide antibiotic ADEP4 activates ClpP protease and the cell's proteolytic machinery, killing persister cells by forcing them to degrade a range of cellular proteins. This is a potentially important result, suggesting that combining compounds such as ADEP4 with conventional antibiotics could provide new and robust strategies for the control of chronic infections.

RNA-guided CRISPR-Cas9 proteins have been widely used for genome editing, but their off-target activities limit broad application. The minimal Cas9 ortholog from Staphylococcus aureus (SaCas9) is commonly used for in vivo genome editing; however, no variant conferring high genome-wide specificity is available. Here, we report rationally engineered SaCas9 variants with highly specific genome-wide activity in human cells without compromising ontarget efficiency. One engineered variant, referred to as SaCas9-HF, dramatically improved genome-wide targeting accuracy based on the genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) method and targeted deep sequencing analyses. Among 15 tested human endogenous sites with the canonical NNGRRT protospacer adjacent motif (PAM), SaCas9-HF rendered no detectable off-target activities at 9 sites, minimal off-target activities at 6 sites, and comparable ontarget efficiencies to those of wild-type SaCas9. Furthermore, among 4 known promiscuous targeting sites, SaCas9-HF profoundly reduced off-target activities compared with wild type. When delivered by an adeno-associated virus vector, SaCas9-HF also showed reduced off-target effects when targeting VEGFA in a human retinal pigmented epithelium cell line compared with wild type. Then, we further altered a previously described variant named KKH-SaCas9 that has a wider PAM recognition range. Similarly, the resulting KKH-HF remarkably reduced off-target activities and increased on- to off-target editing ratios. Our finding provides an alternative to wild-type SaCas9 for genome editing applications requiring exceptional genome-wide precision.

In this review, we chart the major milestones in the research progress on the DyP-type peroxidase family over the past decade. Though mainly distributed among bacteria and fungi, this family actually exhibits more widespread diversity. Advanced tertiary structural analyses have revealed common and different features among members of this family. Notably, the catalytic cycle for the peroxidase activity of DyP-type peroxidases appears to be different from that of other ubiquitous heme peroxidases. DyP-type peroxidases have also been reported to possess activities in addition to peroxidase function, including hydrolase or oxidase activity. They also show various cellular distributions, functioning not only inside cells but also outside of cells. Some are also cargo proteins of encapsulin. Unique, noteworthy functions include a key role in life-cycle switching in Streptomyces and the operation of an iron transport system in Staphylococcus aureus, Bacillus subtilis and Escherichia coli. We also present several probable physiological roles of DyP-type peroxidases that reflect the widespread distribution and function of these enzymes. Lignin degradation is the most common function attributed to DyP-type peroxidases, but their activity is not high compared with that of standard lignin-degrading enzymes. From an environmental standpoint, degradation of natural antifungal anthraquinone compounds is a specific focus of DyP-type peroxidase research. Considered in its totality, the DyP-type peroxidase family offers a rich source of diverse and attractive materials for research scientists.

Metal acquisition is a vital microbial process in metal-scarce environments, such as inside a host. Using metabolomic exploration, targeted mutagenesis, and biochemical analysis, we discovered an operon in Staphylococcus aureus that encodes the different functions required for the biosynthesis and trafficking of a broad-spectrum metallophore related to plant nicotianamine (here called staphylopine). The biosynthesis of staphylopine reveals the association of three enzyme activities: a histidine racemase, an enzyme distantly related to nicotianamine synthase, and a staphylopine dehydrogenase belonging to the DUF2338 family. Staphylopine is involved in nickel, cobalt, zinc, copper, and iron acquisition, depending on the growth conditions. This biosynthetic pathway is conserved across other pathogens, thus underscoring the importance of this metal acquisition strategy in infection.