Cell Wall Precursor Research Articles

The Impact of Growing Area on the Expression of Fruit Traits Related to Sensory Perception in Two Tomato Cultivars.

Environmental conditions greatly influence the quality of tomato fruit by affecting the expression of genes, the abundance of metabolites, and the perception of sensorial attributes. In this study, a fruit transcriptome investigation, a sensory test, and a metabolomic analysis were performed to evaluate the impact of the environment on two popular tomato cultivars grown in two Italian regions. The transcriptional profile of each cultivar, cultivated in two different areas, highlighted differential expression in genes involved in pathways related to cell wall components such as pectin, lignin, and hemicellulose and sugars as well as in amino acids, phenylpropanoids, and pigment synthesis. The cultivation area mainly affects sensory attributes related to texture and flavor and the metabolic pattern of cell wall precursors, sugars, glutamate, aspartate, and carotenoids. In the two genotypes cultivated in the same environment, some attributes and fruit-related quality processes are similarly affected, while others are differently influenced based on the specific genetic makeup of the tomato. A combination of transcriptomic, sensory, and metabolomic data obtained from the two tomato genotypes revealed that the environment has a profound effect on specific sensory traits, providing information on factors that shape the specific characteristics and genetic targets for improving tomato fruit characteristics.

Nitrate assimilation compensates for cell wall biosynthesis in the absence of Aspergillus fumigatus phosphoglucose isomerase.

Phosphoglucose isomerase (PGI) links glycolysis, the pentose phosphate pathway (PPP), and the synthesis of cell wall precursors in fungi by facilitating the reversible conversion between glucose-6-phosphate (Glc6p) and fructose-6-phosphate (Fru6P). In a previous study, we established the essential role of PGI in cell wall biosynthesis in the opportunistic human fungal pathogen Aspergillus fumigatus, highlighting its potential as a therapeutic target. In this study, we conducted transcriptomic analysis and discovered that the Δpgi mutant exhibited enhanced glycolysis, reduced PPP, and an upregulation of cell wall precursor biosynthesis pathways. Phenotypic analysis revealed defective protein N-glycosylation in the mutant, notably the absence of glycosylated virulence factors DPP V and catalase 1. Interestingly, the cell wall defects in the mutant were not accompanied by activation of the MpkA-dependent cell wall integrity (CWI) signaling pathway. Instead, nitrate assimilation was activated in the Δpgi mutant, stimulating glutamine synthesis and providing amino donors for chitin precursor biosynthesis. Blocking the nitrate assimilation pathway severely impaired the growth of the Δpgi mutant, highlighting the crucial role of nitrate assimilation in rescuing cell wall defects. This study unveils the connection between nitrogen assimilation and cell wall compensation in A. fumigatus.IMPORTANCEAspergillus fumigatus is a common and serious human fungal pathogen that causes a variety of diseases. Given the limited availability of antifungal drugs and increasing drug resistance, it is imperative to understand the fungus' survival mechanisms for effective control of fungal infections. Our previous study highlighted the essential role of A. fumigatus PGI in maintaining cell wall integrity, phosphate sugar homeostasis, and virulence. The present study further illuminates the involvement of PGI in protein N-glycosylation. Furthermore, this research reveals that the nitrogen assimilation pathway, rather than the canonical MpkA-dependent CWI pathway, compensates for cell wall deficiencies in the mutant. These findings offer valuable insights into a novel adaptation mechanism of A. fumigatus to address cell wall defects, which could hold promise for the treatment of infections.

Semisynthetic guanidino lipoglycopeptides with potent in vitro and in vivo antibacterial activity.

Gram-positive bacterial infections present a major clinical challenge, with methicillin- and vancomycin-resistant strains continuing to be a cause for concern. In recent years, semisynthetic vancomycin derivatives have been developed to overcome this problem as exemplified by the clinically used telavancin, which exhibits increased antibacterial potency but has also raised toxicity concerns. Thus, glycopeptide antibiotics with enhanced antibacterial activities and improved safety profiles are still necessary. We describe the development of a class of highly potent semisynthetic glycopeptide antibiotics, the guanidino lipoglycopeptides, which contain a positively charged guanidino moiety bearing a variable lipid group. These glycopeptides exhibited enhanced in vitro activity against a panel of Gram-positive bacteria including clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant strains, showed minimal toxicity toward eukaryotic cells, and had a low propensity for resistance selection. Mechanistically, guanidino lipoglycopeptides engaged with bacterial cell wall precursor lipid II with a higher binding affinity than vancomycin. Binding to both wild-type d-Ala-d-Ala lipid II and the vancomycin-resistant d-Ala-d-Lac variant was confirmed, providing insight into the enhanced activity of guanidino lipoglycopeptides against vancomycin-resistant isolates. The in vivo efficacy of guanidino lipoglycopeptide EVG7 was evaluated in a S. aureus murine thigh infection model and a 7-day sepsis survival study, both of which demonstrated superiority to vancomycin. Moreover, the minimal to mild kidney effects at supratherapeutic doses of EVG7 indicate an improved therapeutic safety profile compared with vancomycin. These findings position guanidino lipoglycopeptides as candidates for further development as antibacterial agents for the treatment of clinically relevant multidrug-resistant Gram-positive infections.

Host defence peptide plectasin targets bacterial cell wall precursor lipid II by a calcium-sensitive supramolecular mechanism

Antimicrobial resistance is a leading cause of mortality, calling for the development of new antibiotics. The fungal antibiotic plectasin is a eukaryotic host defence peptide that blocks bacterial cell wall synthesis. Here, using a combination of solid-state nuclear magnetic resonance, atomic force microscopy and activity assays, we show that plectasin uses a calcium-sensitive supramolecular killing mechanism. Efficient and selective binding of the target lipid II, a cell wall precursor with an irreplaceable pyrophosphate, is achieved by the oligomerization of plectasin into dense supra-structures that only form on bacterial membranes that comprise lipid II. Oligomerization and target binding of plectasin are interdependent and are enhanced by the coordination of calcium ions to plectasin’s prominent anionic patch, causing allosteric changes that markedly improve the activity of the antibiotic. Structural knowledge of how host defence peptides impair cell wall synthesis will likely enable the development of superior drug candidates.

Metabolomics unveil key pathways underlying the synergistic activities of aztreonam and avibactam against multidrug-resistant Escherichia coli.

Aztreonam/avibactam is effective against serious infections caused by Gram-negative bacteria including Enterobacterales harboring metallo-β-lactamases. While the utility of this combination has been established in vitro and in clinical trials, the purpose of this study is to enhance our understanding of the underlying mechanism responsible for their activities through metabolomic profiling of a multidrug-resistant Escherichia coli clinical isolate. Metabolomic analyses of time-dependent changes in endogenous bacterial metabolites in a clinical isolate of a multidrug-resistant E. coli treated with aztreonam and avibactam were performed. E. coli metabolomes were compared at 15min, 1h and 24h following treatments with either avibactam (4mg/L), aztreonam (4mg/L), or aztreonam (4mg/L) + avibactam (4mg/L). Drug treatment affected 326 metabolites with magnitude changes of at least 2-fold, most of which are involved primarily in peptidoglycan biosynthesis, nucleotide metabolism, and lipid metabolism.The feedstocks for peptidoglycan synthesis were depleted by aztreonam/avibactam combination;a significant downstream increase in nucleotide metabolites and a release of lipids were observed at the three timepoints. The findings indicate that the aztreonam/avibactam combination accelerates structural damage to the bacterial membrane structure and their actions were immediate and sustained compared to aztreonam or avibactam alone. By inhibiting the production of crucial cell wall precursors, the combination may have inflicted damages on bacterial DNA.

Identification and in vitro characterization of UDP-GlcNAc-RNA cap-modifying and decapping enzymes.

In recent years, several noncanonical RNA caps derived from cofactors and metabolites have been identified. Purine-containing RNA caps have been extensively studied, with multiple decapping enzymes identified and efficient capture and sequencing protocols developed for nicotinamide adenine dinucleotide (NAD)-RNA, which allowed for a stepwise elucidation of capping functions. Despite being identified as an abundant noncanonical RNA-cap, UDP-sugar-capped RNA remains poorly understood, which is partly due to its complex in vitro preparation. Here, we describe a scalable synthesis of sugar-capped uridine-guanosine dinucleotides from readily available protected building blocks and their enzymatic conversion into several cell wall precursor-capped dinucleotides. We employed these capped dinucleotides in T7 RNA polymerase-catalyzed in vitro transcription reactions to efficiently generate RNAs capped with uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), its N-azidoacetyl derivative UDP-GlcNAz, and various cell wall precursors. We furthermore identified four enzymes capable of processing UDP-GlcNAc-capped RNA in vitro: MurA, MurBand MurC from Escherichia coli can sequentially modify the sugar-cap structure and were used to introduce a bioorthogonal, clickable moiety, and the human Nudix hydrolase Nudt5 was shown to efficiently decap UDP-GlcNAc-RNA. Our findings underscore the importance of efficient synthetic methods for capped model RNAs. Additionally, we provide useful enzymatic tools that could be utilized in the development and application of UDP-GlcNAc capture and sequencing protocols. Such protocols are essential for deepening our understanding of the widespread yet enigmatic GlcNAc modification of RNA and its physiological significance.

Natural Antimicrobials in Dairy Products: Benefits, Challenges, and Future Trends.

This review delves into using natural antimicrobials in the dairy industry and examines various sources of these compounds, including microbial, plant, and animal sources. It discusses the mechanisms by which they inhibit microbial growth, for example, by binding to the cell wall's precursor molecule of the target microorganism, consequently inhibiting its biosynthesis, and interfering in the molecule transport mechanism, leading to cell death. In general, they prove to be effective against the main pathogens and spoilage found in food, such as Escherichia coli, Staphylococcus aureus, Bacillus spp., Salmonella spp., mold, and yeast. Moreover, this review explores encapsulation technology as a promising approach for increasing the viability of natural antimicrobials against unfavorable conditions such as pH, temperature, and oxygen exposure. Finally, this review examines the benefits and challenges of using natural antimicrobials in dairy products. While natural antimicrobials offer several advantages, including improved safety, quality, and sensory properties of dairy products, it is crucial to be aware of the challenges associated with their use, such as potential allergenicity, regulatory requirements, and consumer perception. This review concludes by emphasizing the need for further research to identify and develop effective and safe natural antimicrobials for the dairy industry to ensure the quality and safety of dairy products for consumers.

Structural analysis of phosphoribosyltransferase-mediated cell wall precursor synthesis in Mycobacterium tuberculosis.

In Mycobacterium tuberculosis, Rv3806c is a membrane-bound phosphoribosyltransferase (PRTase) involved in cell wall precursor production. It catalyses pentosyl phosphate transfer from phosphoribosyl pyrophosphate to decaprenyl phosphate, to generate 5-phospho-β-ribosyl-1-phosphoryldecaprenol. Despite Rv3806c being an attractive drug target, structural and molecular mechanistic insight into this PRTase is lacking. Here we report cryogenic electron microscopy structures for Rv3806c in the donor- and acceptor-bound states. In a lipidic environment, Rv3806c is trimeric, creating a UbiA-like fold. Each protomer forms two helical bundles, which, alongside the bound lipids, are required for PRTase activity in vitro. Mutational and functional analyses reveal that decaprenyl phosphate and phosphoribosyl pyrophosphate bind the intramembrane and extramembrane cavities of Rv3806c, respectively, in a distinct manner to that of UbiA superfamily enzymes. Our data suggest a model for Rv3806c-catalysed phosphoribose transfer through an inverting mechanism. These findings provide a structural basis for cell wall precursor biosynthesis that could have potential for anti-tuberculosis drug development.

Optimizations of lipid II synthesis: an essential glycolipid precursor in bacterial cell wall synthesis and a validated antibiotic target.

Lipid II is an essential glycolipid found in bacteria. Accessing this valuable cell wall precursor is important both for studying cell wall synthesis and for studying/identifying novel antimicrobial compounds. Herein, we describe optimizations to the modular chemical synthesis of lipid II and unnatural analogues. In particular, the glycosylation step, a critical step in the formation of the central disaccharide unit (GlcNAc-MurNAc), was optimized. This was achieved by employing the use of glycosyl donors with diverse leaving groups. The key advantage of this approach lies in its adaptability, allowing for the generation of a wide array of analogues through the incorporation of alternative building blocks at different stages of synthesis.

Hypothetical protein CuvA (Rv1422) from Mycobacterium tuberculosis H37Rv interacts with uridine diphosphate N-acetylglucosamine as a key precursor of cell wall

Mycobacterium tuberculosis CuvA (Rv1422, MtCuvA) has previously been suggested that it may play a critical role in nutrient utilization and cell wall synthesis required for physiological adaptation in a host cell, but its biochemical details remain unclear. Our previous studies showed that MtCuvA can bind to uridine diphosphate (UDP) sugars as a cell wall precursor component. To verify its functional roles, we report here the biochemical properties of MtCuvA for the binding of UDP-N-acetylglucosamine (GlcNAc) using site-directed mutagenesis and docking simulation. The KD values for UDP-sugars indicate that MtCuvA prefers to bind UDP-GlcNAc as a physiological ligand compared to UDP-glucose. Mutational studies of MtCuvA showed that H12A, T33A, D36A, Q154A, S196, T199A, N226A, and H298A mutants significantly affected the binding to UDP-GlcNAc. We also observed that UDP, but not GlcNAc, could bind to MtCuvA. These results imply that the presence of UDP moiety in the ligand is necessary for interaction with MtCuvA. Moreover, mutational studies of MtCuvA with UDP showed that residues H12, S196, T199, N226, and H298 may be involved in its binding to the UDP moiety, almost consistent with the docking simulation results. Our results provide an insight into the interaction of MtCuvA with UDP-GlcNAc as a key precursor of peptidoglycan.

Tracking Cell Wall-Anchored Proteins in Gram-Positive Bacteria.

Cell wall-anchored surface proteins are integral components of Gram-positive bacterial cell envelope and vital for bacterial survival in different environmental niches. To fulfill their functions, surface protein precursors translocate from cytoplasm to bacterial cell surface in three sequential steps: secretion across the cytoplasmic membrane, covalently anchoring to the cell wall precursor lipid II by sortase A, and incorporation of the lipid II-linked precursors into mature cell wall peptidoglycan. Here, we describe a series of immunofluorescence microscopy methods to track the subcellular localization of cell wall-anchored proteins along the sorting pathway. While the protocols are tailored to Staphylococcus aureus, they can be readily adapted to localize cell wall-anchored proteins as well as membrane proteins in other Gram-positive bacteria.

Nanoparticle‐Based Formulations of Glycopeptide Antibiotics: A Means for Overcoming Vancomycin Resistance in Bacterial Pathogens?

The occurrence and spreading of multidrug resistance in bacterial pathogens represent one of the greatest challenges of the 21st century. With fewer and fewer new antibiotics in development, novel and unconventional concepts in drug development and formulation need to be explored to overcome the global antibiotics crisis. Biomedical nanotechnology offers promising strategies for the reformulation of established antibiotics in such a way that they regain their activity against resistant pathogens. Herein, promising developments aimed at the synthesis of potent nanoparticle‐based formulations of glycopeptide antibiotics are reviewed. While glycopeptide antibiotics such as vancomycin and teicoplanin are widely used as drugs of last resort against Gram‐positive pathogens such as methicillin‐resistant Staphylococcus aureus (MRSA), bacterial resistance against these drugs is spreading. However, inhibiting cell wall synthesis via binding to cell wall precursor peptidoglycans, glycopeptide antibiotics exhibit a unique mode of action that renders them particularly promising candidates for nanoparticle‐based formulations with the ability to overcome glycopeptide antibiotic resistance. Herein, different formulation concepts based on multivalent presentation and encapsulation are introduced, different synthesis and conjugation strategies are discussed, and an attempt is made to rationalize their effects on the antibacterial activity of the resulting nanoparticle formulations.

Disrupting Central Carbon Metabolism Increases β-Lactam Antibiotic Susceptibility in Vibrio cholerae.

Antibiotic tolerance, the ability of bacteria to sustain viability in the presence of typically bactericidal antibiotics for extended time periods, is an understudied contributor to treatment failure. The Gram-negative pathogen Vibrio cholerae, the causative agent of cholera, becomes highly tolerant to β-lactam antibiotics (penicillin and related compounds) in a process requiring the two-component system VxrAB. VxrAB is induced by exposure to cell wall damaging conditions, which results in the differential regulation of >100 genes. While the effectors of VxrAB are relatively well known, VxrAB environment-sensing and activation mechanisms remain a mystery. Here, we used transposon mutagenesis to screen for mutants that spontaneously upregulate VxrAB signaling. This screen was answered by genes known to be required for proper cell envelope homeostasis, validating the approach. Unexpectedly, we also uncovered a new connection between central carbon metabolism and antibiotic tolerance in Vibrio cholerae. Inactivation of pgi (vc0374, coding for glucose-6-phosphate isomerase) resulted in an intracellular accumulation of glucose-6-phosphate and fructose-6-phosphate, concomitant with a marked cell envelope defect, resulting in VxrAB induction. Deletion of pgi also increased sensitivity to β-lactams and conferred a growth defect on salt-free LB, phenotypes that could be suppressed by deleting sugar uptake systems and by supplementing cell wall precursors in the growth medium. Our data suggest an important connection between central metabolism and cell envelope integrity and highlight a potential new target for developing novel antimicrobial agents. IMPORTANCE Antibiotic tolerance (the ability to survive exposure to antibiotics) is a stepping stone toward antibiotic resistance (the ability to grow in the presence of antibiotics), an increasingly common cause of antibiotic treatment failure. The mechanisms promoting tolerance are poorly understood. Here, we identified central carbon metabolism as a key contributor to antibiotic tolerance and resistance. A strain with a mutation in a sugar utilization pathway accumulates metabolites that likely shut down the synthesis of cell wall precursors, which weakens the cell wall and thus increases susceptibility to cell wall-active drugs. Our results illuminate the connection between central carbon metabolism and cell wall homeostasis in V. cholerae and suggest that interfering with metabolism may be a fruitful future strategy for the development of antibiotic adjuvants.

Growth rate is modulated by monitoring cell wall precursors in Bacillus subtilis.

How bacteria link their growth rate to external nutrient conditions is unknown. To investigate how Bacillus subtilis cells alter the rate at which they expand their cell walls as they grow, we compared single-cell growth rates of cells grown under agar pads with the density of moving MreB filaments under a variety of growth conditions. MreB filament density increases proportionally with growth rate. We show that both MreB filament density and growth rate depend on the abundance of Lipid II and murAA, the first gene in the biosynthetic pathway creating the cell wall precursor Lipid II. Lipid II is sensed by the serine/threonine kinase PrkC, which phosphorylates RodZ and other proteins. We show that phosphorylated RodZ increases MreB filament density, which in turn increases cell growth rate. We also show that increasing the activity of this pathway in nutrient-poor media results in cells that elongate faster than wild-type cells, which means that B. subtilis contains spare 'growth capacity'. We conclude that PrkC functions as a cellular rheostat, enabling fine-tuning of cell growth rates in response to Lipid II in different nutrient conditions.

Resistance to the Bacteriocin Lcn972 Deciphered by Genome Sequencing.

In view of the current threat of antibiotic resistance, new antimicrobials with low risk of resistance development are demanded. Lcn972 is a lactococcal bacteriocin that inhibits septum formation by binding to the cell wall precursor lipid II in Lactococcus. It has a species-specific spectrum of activity, making Lcn972 an attractive template to develop or improve existing antibiotics. The aim of this work was to identify mutations present in the Lcn972-resistant clone Lactococcus cremoris D1-20, previously evolved from the sensitive strain L. cremoris MG1614. Whole-genome sequencing and comparison over the reference genome L. cremoris MG1363 identified several unexpected mutations in the parental strain MG1614, likely selected during in-house propagation. In the Lcn972R clone, two previously identified mutations were mapped and confirmed. Additionally, another transposition event deregulating cellobiose uptake was identified along with three point mutations of unknown consequences for Lcn972 resistance. Two new independent evolution experiments exposing L. cremoris MG1614 to Lcn972 revealed transposition of IS981 into the LLMG_RS12285 locus as the predominant mutation selected by Lcn972. This event occurs early during evolution and was found in 100% of the evolved clones, while other mutations were not selected. Therefore, activation of LLMG_RS12285 coding for a putative anti-ECF (extra-cytoplasmic function) sigma factor is regarded as the main Lcn972 resistance factor in L. cremoris MG1614.

Plasma Membrane-Cell Wall Feedback in Bacteria.

Most bacteria have cell wall peptidoglycan surrounding their plasma membranes. The essential cell wall provides a scaffold for the envelope, protection against turgor pressure and is a proven drug target. Synthesis of the cell wall involves reactions that span cytoplasmic and periplasmic compartments. Bacteria carry out the last steps of cell wall synthesis along their plasma membrane. The plasma membrane in bacteria is heterogeneous and contains membrane compartments. Here, I outline findings that highlight the emerging notion that plasma membrane compartments and the cell wall peptidoglycan are functionally intertwined. I start by providing models of cell wall synthesis compartmentalization within the plasma membrane in mycobacteria, Escherichia coli, and Bacillus subtilis. Then, I revisit literature that supports a role for the plasma membrane and its lipids in modulating enzymatic reactions that synthesize cell wall precursors. I also elaborate on what is known about bacterial lateral organization of the plasma membrane and the mechanisms by which organization is established and maintained. Finally, I discuss the implications of cell wall partitioning in bacteria and highlight how targeting plasma membrane compartmentalization serves as a way to disrupt cell wall synthesis in diverse species.

Polar protein Wag31 both activates and inhibits cell wall metabolism at the poles and septum.

Mycobacterial cell elongation occurs at the cell poles; however, it is not clear how cell wall insertion is restricted to the pole or how it is organized. Wag31 is a pole-localized cytoplasmic protein that is essential for polar growth, but its molecular function has not been described. In this study we used alanine scanning mutagenesis to identify Wag31 residues involved in cell morphogenesis. Our data show that Wag31 helps to control proper septation as well as new and old pole elongation. We have identified key amino acid residues involved in these essential functions. Enzyme assays revealed that Wag31 interacts with lipid metabolism by modulating acyl-CoA carboxylase (ACCase) activity. We show that Wag31 does not control polar growth by regulating the localization of cell wall precursor enzymes to the Intracellular Membrane Domain, and we also demonstrate that phosphorylation of Wag31 does not substantively regulate peptidoglycan metabolism. This work establishes new regulatory functions of Wag31 in the mycobacterial cell cycle and clarifies the need for new molecular models of Wag31 function.

Targeting membrane-bound bacterial cell wall precursors: a tried and true antibiotic strategy in nature and the clinic.

Since Fleming's discovery of penicillin nearly a century ago, a bounty of natural product antibiotics have been discovered, many of which continue to be of clinical importance today. The structural diversity encountered among nature's repertoire of antibiotics is mirrored by the varying mechanisms of action by which they selectively target and kill bacterial cells. The ability for bacteria to construct and maintain a strong cell wall is essential for their robust growth and survival under a range of conditions. However, the need to maintain the cell wall also presents a vulnerability that is exploited by many natural antibiotics. Bacterial cell wall biosynthesis involves both the construction of complex membrane-bound precursor molecules and their subsequent crosslinking by dedicated enzymes. Interestingly, many naturally occurring antibiotics function not by directly inhibiting the enzymes associated with cell wall biosynthesis, but rather by binding tightly to their membrane-bound substrates. Such substrate sequestration mechanisms are comparatively rare outside of the antibiotics space with most small-molecule drug discovery programs instead aimed at developing inhibitors of target enzymes. In this feature article we provide the reader with an overview of the unique and ever increasing family of natural product antibiotics known to specifically function by binding to membrane-anchored bacterial cell wall precursors. In doing so, we highlight both our own contributions to the field as well as those made by other researchers engaged in exploring the potential offered by antibiotics that target bacterial cell wall precursors.

Strategies and tactics for the synthesis of lipid I and II and shortened analogues: functional building blocks of bacterial cell wall biosynthesis.

Covering: the literature up to 2022This study discusses various synthetic strategies for the synthesis of lipid II, the pivotal bacterial cell wall precursor. In detail, it examines different solution phase approaches, reviews various solid phase sequences, and evaluates enzymatic ventures. The underlying rationale, scope, limitations, and perspectives of these strategies are discussed. The focus is on the tactics and strategies towards the authentic peptidoglycan compound, as well as analogues thereof with shortened side chains, which are increasingly recognized as more beneficial surrogates with more favorable physicochemical properties.

Evaluation of analogs of mutacin 1140 in systemic and cutaneous methicillin-resistant Staphylococcus aureus infection models in mice.

Mutacin 1140 (Mu1140) is a potent antibiotic against Gram-positive bacteria, such as Staphylococcus aureus. The antibiotic is produced by the oral bacterium Streptococcus mutans and is a member of the epidermin family of type AI lantibiotics. The antibiotic exerts its inhibitory activity by binding to the cell wall precursor lipid II, blocking cell wall synthesis, and by disrupting bacterial membranes. In previous studies, the novel K2A and R13A analogs of Mu1140 have been identified to have superior pharmacokinetic properties compared to native Mu1140. In this study, the use of a combined formulation of the Mu1140 K2A and R13A analogs was shown to be more effective at treating MRSA bacteremia than the native Mu1140 or vancomycin. The analogs were also shown to be effective in treating an MRSA skin infection. The use of K2A and R13A analogs may provide a future alternative for treating serious Gram-positive bacterial infections. In a previous study, the Mu1140 analogs were shown to have significantly longer drug clearance times, leading to higher plasma concentrations over time. These properties warranted further testing to determine whether the analogs are effective for the treatment of systemic MRSA and acute skin infections. In this study, Mu1140 analogs were shown to be more effective than currently available treatments for systemic and skin MRSA infections. Further, the study clearly shows that the new analogs are superior to native Mu1140 for the treatment of a systemic MRSA infection. These findings support continued drug product development efforts using the K2A and R13A Mu1140 analogs, and that these analogs may ameliorate the outcome of serious bacterial infections.