Post-transcriptional control of the essential enzyme MurF by a small regulatory RNA in Brucella abortus.

Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation of gene expression through the use of small regulatory RNAs (sRNAs). Here, we characterize a Brucella sRNAs called MavR (for MurF- and virulence-regulating sRNA), and we demonstrate that MavR is required for the full virulence of B. abortus in macrophages and in a mouse model of chronic infection. Transcriptomic and proteomic studies revealed that a major regulatory target of MavR is MurF. MurF is an essential protein that catalyzes the final cytoplasmic step in peptidoglycan (PG) synthesis; however, we did not detect any differences in the amount or chemical composition of PG in the ΔmavR mutant. A 6-nucleotide regulatory seed region within MavR was identified, and mutation of this seed region resulted in dysregulation of MurF production, as well as significant attenuation of infection in a mouse model. Overall, the present study underscores the importance of sRNA regulation in the physiology and virulence of Brucella.

Staphylococcus aureus resistance to albocycline can be achieved by mutations that alter cellular NAD/PH pools.

Small molecule target identification is a critical step in modern antibacterial drug discovery, particularly against multi-drug resistant pathogens. Albocycline (ALB) is a macrolactone natural product with potent activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) whose mechanism of action has been elusive to date. Herein, we report biochemical and genomic studies that reveal ALB does not target bacterial peptidoglycan biosynthesis or the ribosome; rather, it appears to modulate NADPH ratios and upregulate redox sensing in the cell consistent with previous studies at Upjohn. Owing to the complexity inherent in biological pathways, further genomic assays are needed to identify the true molecular target(s) of albocycline.

Synthesis and biological evaluation of semi-synthetic albocycline analogs.

Albocycline (ALB) is a unique macrolactone natural product with potent, narrow-spectrum activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate (VISA), and vancomycin-resistant S. aureus (VRSA) strains (MIC = 0.5-1.0 µg/mL). Described herein is the synthesis and evaluation of a novel series analogs derived from albocycline by functionalization at three specific sites: the C2-C3 enone, the tertiary carbinol at C4, and the allylic C16 methyl group. Exploration of the structure-activity relationships (SAR) by means of minimum inhibitory concentration assays (MICs) revealed that C4 ester analog 6 was twice as potent as ALB, which represents a class of lead compound that can be further studied to address multi-drug resistant pathogens.

A Novel, Broad-Spectrum Antimicrobial Combination for the Treatment of Pseudomonas aeruginosa Corneal Infections.

Bacterial keratitis causes significant blindness, yet antimicrobial resistance has rendered current treatments ineffective. Polymyxin B-trimethoprim (PT) plus rifampin has potent in vitro activity against Staphylococcus aureus and Pseudomonas aeruginosa, two important causes of keratitis. Here we further characterize this combination against P. aeruginosa in a murine keratitis model. PT plus rifampin performed comparably to or better than moxifloxacin, the gold standard, suggesting that the combination may be a promising therapy for bacterial keratitis.

Development of a Broad-Spectrum Antimicrobial Combination for the Treatment of Staphylococcus aureus and Pseudomonas aeruginosa Corneal Infections.

Staphylococcus aureus and Pseudomonas aeruginosa are two of the most common causes of bacterial keratitis and corresponding corneal blindness. Accordingly, such infections are predominantly treated with broad-spectrum fluoroquinolones, such as moxifloxacin. Yet, the rising fluoroquinolone resistance has necessitated the development of alternative therapeutic options. Herein, we describe the development of a polymyxin B-trimethoprim (PT) ophthalmic formulation containing the antibiotic rifampin, which exhibits synergistic antimicrobial activity toward a panel of contemporary ocular clinical S. aureus and P. aeruginosa isolates, low spontaneous resistance frequency, and in vitro bactericidal kinetics and antibiofilm activities equaling or exceeding the antimicrobial properties of moxifloxacin. The PT plus rifampin combination also demonstrated increased efficacy in comparison to those of either commercial PT or moxifloxacin in a murine keratitis model of infection, resulting in bacterial clearance of 70% in the animals treated. These results suggest that the combination of PT and rifampin may represent a novel antimicrobial agent in the treatment of bacterial keratitis.

Characterizing the Antimicrobial Properties of 405 nm Light and the Corning® Light-Diffusing Fiber Delivery System.

BACKGROUND AND OBJECTIVES: Hospital-acquired infections (HAIs) and multidrug resistant bacteria pose a significant threat to the U.S. healthcare system. With a dearth of new antibiotic approvals, novel antimicrobial strategies are required to help solve this problem. Violet-blue visible light (400-470 nm) has been shown to elicit strong antimicrobial effects toward many pathogens, including representatives of the ESKAPE bacterial pathogens, which have a high propensity to cause HAIs. However, phototherapeutic solutions to prevention or treating infections are currently limited by efficient and nonobtrusive light-delivery mechanisms. STUDY DESIGN/MATERIALS AND METHODS: Here, we investigate the in vitro antimicrobial properties of flexible Corning® light-diffusing fiber (LDF) toward members of the ESKAPE pathogens in a variety of growth states and in the context of biological materials. Bacteria were grown on agar surfaces, in liquid culture and on abiotic surfaces. We also explored the effects of 405 nm light within the presence of lung surfactant, human serum, and on eukaryotic cells. Pathogens tested include Enterococcus spp, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., Staphylococcus epidermidis, Streptococcus pyogenes, Candida albicans, and Escherichia coli. RESULTS: Overall, the LDF delivery of 405 nm violet-blue light exerted a significant degree of microbicidal activity against a wide range of pathogens under diverse experimental conditions. CONCLUSIONS: The results exemplify the fiber's promise as a non-traditional approach for the prevention and/or therapeutic intervention of HAIs. Lasers Surg. Med. © 2019 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.

Endoribonuclease YbeY Is Linked to Proper Cellular Morphology and Virulence in Brucella abortus.

The YbeY endoribonuclease is one of the best-conserved proteins across the kingdoms of life. In the present study, we demonstrated that YbeY in Brucella abortus is linked to a variety of important activities, including proper cellular morphology, mRNA transcript levels, and virulence. Deletion of ybeY in B. abortus led to a small-colony phenotype when the bacteria were grown on agar medium, as well as to significant aberrations in the morphology of the bacterial cell as evidenced by electron microscopy. Additionally, compared to the parental strain, the ΔybeY strain was significantly attenuated in both macrophage and mouse models of infection. The ΔybeY strain also showed increased sensitivities to several in vitro-applied stressors, including bile acid, hydrogen peroxide, SDS, and paraquat. Transcriptomic analysis revealed that a multitude of mRNA transcripts are dysregulated in the ΔybeY strain, and many of the identified mRNAs encode proteins involved in metabolism, nutrient transport, transcriptional regulation, and flagellum synthesis. We subsequently constructed gene deletion strains of the most highly dysregulated systems, and several of the YbeY-linked gene deletion strains exhibited defects in the ability of the bacteria to survive and replicate in primary murine macrophages. Taken together, these data establish a clear role for YbeY in the biology and virulence of Brucella; moreover, this work further illuminates the highly varied roles of this widely conserved endoribonuclease in bacteria.IMPORTANCEBrucella spp. are highly efficient bacterial pathogens of animals and humans, causing significant morbidity and economic loss worldwide, and relapse of disease often occurs following antibiotic treatment of human brucellosis. As such, novel therapeutic strategies to combat Brucella infections are needed. Ribonucleases in the brucellae are understudied, and these enzymes represent elements that may be potential targets for future treatment approaches. The present work demonstrates the importance of the YbeY endoribonuclease for cellular morphology, efficient control of mRNA levels, and virulence in B. abortus Overall, the results of this study advance our understanding of the critical roles of YbeY in the pathogenesis of the intracellular brucellae and expand our understanding of this highly conserved RNase.

Novel inhibitors of Staphylococcus aureus RnpA that synergize with mupirocin.

We recently discovered RnpA as a promising new drug discovery target for methicillin-resistant S. aureus (MRSA). RnpA is an essential protein that is thought to perform two required cellular processes. As part of the RNA degrasome Rnpa mediates RNA degradation. In combination with rnpB it forms RNase P haloenzymes which are required for tRNA maturation. A high throughput screen identified RNPA2000 as an inhibitor of both RnpA-associated activities that displayed antibacterial activity against clinically relevant strains of S. aureus, including MRSA. Structure-activity studies aimed at improving potency and replacing the potentially metabotoxic furan moiety led to the identification of a number of more potent analogs. Many of these new analogs possessed overt cellular toxicity that precluded their use as antibiotics but two derivatives, including compound 5o, displayed an impressive synergy with mupirocin, an antibiotic used for decolonizing MSRA whose effectiveness has recently been jeopardized by bacterial resistance. Based on our results, compounds like 5o may ultimately find use in resensitizing mupirocin-resistant bacteria to mupirocin.

Elucidating the inhibition of peptidoglycan biosynthesis in Staphylococcus aureus by albocycline, a macrolactone isolated from Streptomyces maizeus.

Antibiotic resistance is a serious threat to global public health, and methicillin-resistant Staphylococcus aureus (MRSA) is a poignant example. The macrolactone natural product albocycline, derived from various Streptomyces strains, was recently identified as a promising antibiotic candidate for the treatment of both MRSA and vancomycin-resistant S. aureus (VRSA), which is another clinically relevant and antibiotic resistant strain. Moreover, it was hypothesized that albocycline's antimicrobial activity was derived from the inhibition of peptidoglycan (i.e., bacterial cell wall) biosynthesis. Herein, preliminary mechanistic studies are performed to test the hypothesis that albocycline inhibits MurA, the enzyme that catalyzes the first step of peptidoglycan biosynthesis, using a combination of biological assays alongside molecular modeling and simulation studies. Computational modeling suggests albocycline exists as two conformations in solution, and computational docking of these conformations to an ensemble of simulated receptor structures correctly predicted preferential binding to S. aureus MurA-the enzyme that catalyzes the first step of peptidoglycan biosynthesis-over Escherichia coli (E. coli) MurA. Albocycline isolated from the producing organism (Streptomyces maizeus) weakly inhibited S. aureus MurA (IC50 of 480 µM) but did not inhibit E. coli MurA. The antimicrobial activity of albocycline against resistant S. aureus strains was superior to that of vancomycin, preferentially inhibiting Gram-positive organisms. Albocycline was not toxic to human HepG2 cells in MTT assays. While these studies demonstrate that albocycline is a promising lead candidate against resistant S. aureus, taken together they suggest that MurA is not the primary target, and further work is necessary to identify the major biological target.

Bacterial Hypoxic Responses Revealed as Critical Determinants of the Host-Pathogen Outcome by TnSeq Analysis of Staphylococcus aureus Invasive Infection.

Staphylococcus aureus is capable of infecting nearly every organ in the human body. In order to infiltrate and thrive in such diverse host tissues, staphylococci must possess remarkable flexibility in both metabolic and virulence programs. To investigate the genetic requirements for bacterial survival during invasive infection, we performed a transposon sequencing (TnSeq) analysis of S. aureus during experimental osteomyelitis. TnSeq identified 65 genes essential for staphylococcal survival in infected bone and an additional 148 mutants with compromised fitness in vivo. Among the loci essential for in vivo survival was SrrAB, a staphylococcal two-component system previously reported to coordinate hypoxic and nitrosative stress responses in vitro. Healthy bone is intrinsically hypoxic, and intravital oxygen monitoring revealed further decreases in skeletal oxygen concentrations upon S. aureus infection. The fitness of an srrAB mutant during osteomyelitis was significantly increased by depletion of neutrophils, suggesting that neutrophils impose hypoxic and/or nitrosative stresses on invading bacteria. To more globally evaluate staphylococcal responses to changing oxygenation, we examined quorum sensing and virulence factor production in staphylococci grown under aerobic or hypoxic conditions. Hypoxic growth resulted in a profound increase in quorum sensing-dependent toxin production, and a concomitant increase in cytotoxicity toward mammalian cells. Moreover, aerobic growth limited quorum sensing and cytotoxicity in an SrrAB-dependent manner, suggesting a mechanism by which S. aureus modulates quorum sensing and toxin production in response to environmental oxygenation. Collectively, our results demonstrate that bacterial hypoxic responses are key determinants of the staphylococcal-host interaction.

A LysR-family transcriptional regulator required for virulence in Brucella abortus is highly conserved among the α-proteobacteria.

Small RNAs are principal elements of bacterial gene regulation and physiology. Two small RNAs in Brucella abortus, AbcR1 and AbcR2, are required for wild-type virulence. Examination of the abcR loci revealed the presence of a gene encoding a LysR-type transcriptional regulator flanking abcR2 on chromosome 1. Deletion of this lysR gene (bab1_1517) resulted in the complete loss of abcR2 expression while no difference in abcR1 expression was observed. The B. abortus bab1_1517 mutant strain was significantly attenuated in macrophages and mice, and bab1_1517 was subsequently named vtlR for virulence-associated transcriptional LysR-family regulator. Microarray analysis revealed three additional genes encoding small hypothetical proteins also under the control of VtlR. Electrophoretic mobility shift assays demonstrated that VtlR binds directly to the promoter regions of abcR2 and the three hypothetical protein-encoding genes, and DNase I footprint analysis identified the specific nucleotide sequence in these promoters that VtlR binds to and drives gene expression. Strikingly, orthologs of VtlR are encoded in a wide range of host-associated α-proteobacteria, and it is likely that the VtlR genetic system represents a common regulatory circuit critical for host-bacterium interactions.

Rot is a key regulator of Staphylococcus aureus biofilm formation.

Staphylococcus aureus is a significant cause of chronic biofilm infections on medical implants. We investigated the biofilm regulatory cascade and discovered that the repressor of toxins (Rot) is part of this pathway. A USA300 community-associated methicillin-resistant S. aureus strain deficient in Rot was unable to form a biofilm using multiple different assays, and we found rot mutants in other strain lineages were also biofilm deficient. By performing a global analysis of transcripts and protein production controlled by Rot, we observed that all the secreted protease genes were up-regulated in a rot mutant, and we hypothesized that this regulation could be responsible for the biofilm phenotype. To investigate this question, we determined that Rot bound to the protease promoters, and we observed that activity levels of these enzymes, in particular the cysteine proteases, were increased in a rot mutant. By inactivating these proteases, biofilm capacity was restored to the mutant, demonstrating they are responsible for the biofilm negative phenotype. Finally, we tested the rot mutant in a mouse catheter model of biofilm infection and observed a significant reduction in biofilm burden. Thus S. aureus uses the transcription factor Rot to repress secreted protease levels in order to build a biofilm.

Neomycin Sulfate Improves the Antimicrobial Activity of Mupirocin-Based Antibacterial Ointments.

In the midst of the current antimicrobial pipeline void, alternative approaches are needed to reduce the incidence of infection and decrease reliance on last-resort antibiotics for the therapeutic intervention of bacterial pathogens. In that regard, mupirocin ointment-based decolonization and wound maintenance practices have proven effective in reducing Staphylococcus aureus transmission and mitigating invasive disease. However, the emergence of mupirocin-resistant strains has compromised the agent's efficacy, necessitating new strategies for the prevention of staphylococcal infections. Herein, we set out to improve the performance of mupirocin-based ointments. A screen of a Food and Drug Administration (FDA)-approved drug library revealed that the antibiotic neomycin sulfate potentiates the antimicrobial activity of mupirocin, whereas other library antibiotics did not. Preliminary mechanism of action studies indicate that neomycin's potentiating activity may be mediated by inhibition of the organism's RNase P function, an enzyme that is believed to participate in the tRNA processing pathway immediately upstream of the primary target of mupirocin. The improved antimicrobial activity of neomycin and mupirocin was maintained in ointment formulations and reduced S. aureus bacterial burden in murine models of nasal colonization and wound site infections. Combination therapy improved upon the effects of either agent alone and was effective in the treatment of contemporary methicillin-susceptible, methicillin-resistant, and high-level mupirocin-resistant S. aureus strains. From these perspectives, combination mupirocin-and-neomycin ointments appear to be superior to that of mupirocin alone and warrant further development.

Small-molecule inhibitors of Staphylococcus aureus RnpA-mediated RNA turnover and tRNA processing.

New agents are urgently needed for the therapeutic treatment of Staphylococcus aureus infections. In that regard, S. aureus RNase RnpA may represent a promising novel dual-function antimicrobial target that participates in two essential cellular processes, RNA degradation and tRNA maturation. Accordingly, we previously used a high-throughput screen to identify small-molecule inhibitors of the RNA-degrading activity of the enzyme and showed that the RnpA inhibitor RNPA1000 is an attractive antimicrobial development candidate. In this study, we used a series of in vitro and cellular assays to characterize a second RnpA inhibitor, RNPA2000, which was identified in our initial screening campaign and is structurally distinct from RNPA1000. In doing so, it was found that S. aureus RnpA does indeed participate in 5'-precursor tRNA processing, as was previously hypothesized. Further, we show that RNPA2000 is a bactericidal agent that inhibits both RnpA-associated RNA degradation and tRNA maturation activities both in vitro and within S. aureus. The compound appears to display specificity for RnpA, as it did not significantly affect the in vitro activities of unrelated bacterial or eukaryotic ribonucleases and did not display measurable human cytotoxicity. Finally, we show that RNPA2000 exhibits antimicrobial activity and inhibits tRNA processing in efflux-deficient Gram-negative pathogens. Taken together, these data support the targeting of RnpA for antimicrobial development purposes, establish that small-molecule inhibitors of both of the functions of the enzyme can be identified, and lend evidence that RnpA inhibitors may have broad-spectrum antimicrobial activities.

Staphylococcus epidermidis agr quorum-sensing system: signal identification, cross talk, and importance in colonization.

Staphylococcus epidermidis is an opportunistic pathogen that is one of the leading causes of medical device infections. Global regulators like the agr quorum-sensing system in this pathogen have received a limited amount of attention, leaving important questions unanswered. There are three agr types in S. epidermidis strains, but only one of the autoinducing peptide (AIP) signals has been identified (AIP-I), and cross talk between agr systems has not been tested. We structurally characterized all three AIP types using mass spectrometry and discovered that the AIP-II and AIP-III signals are 12 residues in length, making them the largest staphylococcal AIPs identified to date. S. epidermidis agr reporter strains were developed for each system, and we determined that cross-inhibitory interactions occur between the agr type I and II systems and between the agr type I and III systems. In contrast, no cross talk was observed between the type II and III systems. To further understand the outputs of the S. epidermidis agr system, an RNAIII mutant was constructed, and microarray studies revealed that exoenzymes (Ecp protease and Geh lipase) and low-molecular-weight toxins were downregulated in the mutant. Follow-up analysis of Ecp confirmed the RNAIII is required to induce protease activity and that agr cross talk modulates Ecp activity in a manner that mirrors the agr reporter results. Finally, we demonstrated that the agr system enhances skin colonization by S. epidermidis using a porcine model. This work expands our knowledge of S. epidermidis agr system function and will aid future studies on cell-cell communication in this important opportunistic pathogen.

Identification of Acinetobacter baumannii serum-associated antibiotic efflux pump inhibitors.

Adaptive antibiotic resistance is a newly described phenomenon by which Acinetobacter baumannii induces efflux pump activity in response to host-associated environmental cues that may, in part, account for antibiotic treatment failures against clinically defined susceptible strains. To that end, during adaptation to growth in human serum, the organism induces approximately 22 putative efflux-associated genes and displays efflux-mediated minocycline tolerance at antibiotic concentrations corresponding to patient serum levels. Here, we show that in addition to minocycline, growth in human serum elicits A. baumannii efflux-mediated tolerance to the antibiotics ciprofloxacin, meropenem, tetracycline, and tigecycline. Moreover, using a whole-cell high-throughput screen and secondary assays, we identified novel serum-associated antibiotic efflux inhibitors that potentiated the activities of antibiotics toward serum-grown A. baumannii. Two compounds, Acinetobacter baumannii efflux pump inhibitor 1 (ABEPI1) [(E)-4-((4-chlorobenzylidene)amino)benezenesulfonamide] and ABEPI2 [N-tert-butyl-2-(1-tert-butyltetrazol-5-yl)sulfanylacetamide], were shown to lead to minocycline accumulation within A. baumannii during serum growth and inhibit the efflux potential of the organism. While both compounds also inhibited the antibiotic efflux properties of the bacterial pathogen Pseudomonas aeruginosa, they did not display significant cytotoxicity toward human cells or mammalian Ca(2+) channel inhibitory effects, suggesting that ABEPI1 and ABEPI2 represent promising structural scaffolds for the development of new classes of bacterial antibiotic efflux pump inhibitors that can be used to potentiate the activities of current and future antibiotics for the therapeutic intervention of Gram-negative bacterial infections.

Genetic pathway in acquisition and loss of vancomycin resistance in a methicillin resistant Staphylococcus aureus (MRSA) strain of clonal type USA300.

An isolate of the methicillin-resistant Staphylococcus aureus (MRSA) clone USA300 with reduced susceptibility to vancomycin (SG-R) (i.e, vancomycin-intermediate S. aureus, VISA) and its susceptible "parental" strain (SG-S) were recovered from a patient at the end and at the beginning of an unsuccessful vancomycin therapy. The VISA phenotype was unstable in vitro generating a susceptible revertant strain (SG-rev). The availability of these 3 isogenic strains allowed us to explore genetic correlates of antibiotic resistance as it emerged in vivo. Compared to the susceptible isolate, both the VISA and revertant strains carried the same point mutations in yycH, vraG, yvqF and lspA genes and a substantial deletion within an intergenic region. The revertant strain carried a single additional frameshift mutation in vraS which is part of two component regulatory system VraSR. VISA isolate SG-R showed complex alterations in phenotype: decreased susceptibility to other antibiotics, slow autolysis, abnormal cell division and increased thickness of cell wall. There was also altered expression of 239 genes including down-regulation of major virulence determinants. All phenotypic properties and gene expression profile returned to parental levels in the revertant strain. Introduction of wild type yvqF on a multicopy plasmid into the VISA strain caused loss of resistance along with loss of all the associated phenotypic changes. Introduction of the wild type vraSR into the revertant strain caused recovery of VISA type resistance. The yvqF/vraSR operon seems to function as an on/off switch: mutation in yvqF in strain SG-R turns on the vraSR system, which leads to increase in vancomycin resistance and down-regulation of virulence determinants. Mutation in vraS in the revertant strain turns off this regulatory system accompanied by loss of resistance and normal expression of virulence genes. Down-regulation of virulence genes may provide VISA strains with a "stealth" strategy to evade detection by the host immune system.

Efficacy of a Novel Antibiotic Drug Combination Toward Multidrug-Resistant Ocular Pathogens.

PURPOSE: Antimicrobial resistance is a global health threat, compounded by the reduction in the discovery of new antibiotics. A repurposed drugs-based approach could provide a viable alternative for the treatment of multidrug-resistant (MDR) bacterial infections. In this study, we sought to evaluate the in vitro efficacy of a novel drug combination, polymyxin B/trimethoprim (PT) + rifampin on MDR isolates from patients with bacterial keratitis in India. METHODS: Forty-three isolates, which included 20 Staphylococcus aureus , 19 Pseudomonas aeruginosa , 3 Pseudomonas stutzeri , and 1 Acinetobacter baumannii , were evaluated for their antibiotic resistance by minimum inhibitory concentration (MIC). Fractional Inhibitory Concentration Index (FICI) testing was performed to measure the antimicrobial impact of PT + rifampin in combination. RESULTS: Among S. aureus isolates, 100% were resistant to at least 1 antibiotic class, 12 (60%) were MDR, and 14 (70%) were classified as methicillin-resistant. Among the gram-negative isolates, >90% were classified as MDR. Fractional Inhibitory Concentration (FIC) testing revealed that PT + rifampin was effective in completely inhibiting growth of all isolates while also displaying additive or synergistic activity in approximately 70% of the strains. Mean FICI values were 0.753 ± 0.311 and 0.791 ± 0.369 for S. aureus and gram-negative isolates, respectively, and a >2-fold reduction in MIC was measured for both PT and rifampin when tested in combination versus alone. CONCLUSIONS: Our data demonstrate the ability of PT + rifampin to eliminate all isolates tested, even those conferring MDR, highlighting the promise of this drug combination for the treatment of bacterial keratitis.

Development of phenyl-urea-based small molecules that target penicillin-binding protein 4.

Staphylococcus aureus has the ability to invade cortical bone osteocyte lacuno-canalicular networks (OLCNs) and cause osteomyelitis. It was recently established that the cell wall transpeptidase, penicillin-binding protein 4 (PBP4), is crucial for this function, with pbp4 deletion strains unable to invade OLCNs and cause bone pathogenesis in a murine model of S. aureus osteomyelitis. Moreover, PBP4 has recently been found to modulate S. aureus resistance to ß-lactam antibiotics. As such, small molecule inhibitors of S. aureus PBP4 may represent dual functional antimicrobial agents that limit osteomyelitis and/or reverse antibiotic resistance. A high throughput screen recently revealed that the phenyl-urea 1 targets PBP4. Herein, we describe a structure-activity relationship (SAR) study on 1. Leveraging in silico docking and modeling, a set of analogs was synthesized and assessed for PBP4 inhibitory activities. Results revealed a preliminary SAR and identified lead compounds with enhanced binding to PBP4, more potent antibiotic resistance reversal, and diminished PBP4 cell wall transpeptidase activity in comparison to 1.

Characterization of the effects of an rpoC mutation that confers resistance to the Fst peptide toxin-antitoxin system toxin.

Overexpression of the Fst toxin in Enterococcus faecalis strain OG1X leads to defects in chromosome segregation, cell division and, eventually, membrane integrity. The M7 mutant derivative of OG1X is resistant to most of these effects but shows a slight growth defect in the absence of Fst. Full-genome sequencing revealed two differences between M7 and its OG1X parent. First, OG1X contains a frameshift mutation that inactivates the etaR response regulator gene, while M7 is a wild-type revertant for etaR. Second, the M7 mutant contains a missense mutation in the rpoC gene, which encodes the ß' subunit of RNA polymerase. Mutagenesis experiments revealed that the rpoC mutation was primarily responsible for the resistance phenotype. Microarray analysis revealed that a number of transporters were induced in OG1X when Fst was overexpressed. These transporters were not induced in M7 in response to Fst, and further experiments indicated that this had a direct protective effect on the mutant cells. Therefore, exposure of cells to Fst appears to have a cascading effect, first causing membrane stress and then potentiation of these effects by overexpression of certain transporters.