SK-03-92 Drug Kills Intracellular Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis affects millions of people worldwide. The emergence of drug-resistant Mycobacterium tuberculosis strains has made treatment more difficult. A drug discovery project initiated to screen natural products identified a lead stilbene compound, and structure function analysis of hundreds of analogs led to the discovery of the SK-03-92 stilbene lead compound with activity against several non-tuberculoid mycobacteria. METHODS: For this study, an MIC analysis and intracellular killing assay were performed to test SK-03-92 against M. tuberculosis grown in vitro as well as within murine macrophage cells. RESULTS: The MIC analysis showed that SK-03-92 had activity against M. tuberculosis in the range of 0.39 to 6.25 µg/mL, including activity against single-drug-resistant strains. Further, an intracellular kill assay demonstrated that the SK-03-92 lead compound killed M. tuberculosis cells within murine macrophage cells. CONCLUSION: Together, the data show the SK-03-92 lead compound can kill M. tuberculosis bacteria within mammalian macrophages.

Proline Transport and Growth Changes in Proline Transport Mutants of Staphylococcus aureus.

Staphylococcus aureus is a major cause of skin/soft tissue infections and more serious infections in humans. The species usually requires the importation of proline to be able to survive. Previous work has shown that single mutations in genes that encode for proline transporters affect the ability of S. aureus to survive in vitro and in vivo. To better understand proline transport in S. aureus, double and triple gene mutant strains were created that targeted the opuD, proP, and putP genes. Single gene mutants had some effect on proline transport, whereas double mutants exhibited significantly lower proline transport. An opuD prop putP triple gene mutant displayed the lowest proline transport under low- and high-affinity conditions. To assess growth differences caused by the mutations, the same mutants were grown in brain heart infusion (BHI) broth and defined staphylococcal medium (DSM) with various concentrations of proline. The triple mutant did not grow in DSM with a low concentration of proline and grew poorly in both DSM with a high proline concentration and BHI broth. These results show that S. aureus has multiple mechanisms to import proline into the cell and knocking out three of the main proline transporters significantly hinders S. aureus growth.

Regulation of Escherichia coli fim gene transcription by GadE and other acid tolerance gene products.

Uropathogenic Escherichia coli (UPEC) cause millions of urinary tract infections each year in the United States. Type 1 pili are important for adherence of UPEC to uroepithelial cells in the human and murine urinary tracts where osmolality and pH vary. Previous work has shown that an acidic pH adversely affects the expression of type 1 pili. To determine if acid tolerance gene products may be regulating E. coli fim gene expression, a bank of K-12 strain acid tolerance gene mutants were screened using fimA-lux, fimB-lux, and fimE-lux fusions on single copy number plasmids. We have determined that a mutation in gadE increased transcription of all three fim genes, suggesting that GadE may be acting as a repressor in a low pH environment. Complementation of the gadE mutation restored fim gene transcription to wild-type levels. Moreover, mutations in gadX, gadW, crp, and cya also affected transcription of the three fim genes. To verify the role GadE plays in type 1 pilus expression, the NU149 gadE UPEC strain was tested. The gadE mutant had higher fimE gene transcript levels, a higher frequency of Phase-OFF positioning of fimS, and hemagglutination titres that were lower in strain NU149 gadE cultured in low pH medium as compared to the wild-type bacteria. The data demonstrate that UPEC fim genes are regulated directly or indirectly by the GadE protein and this could have some future bearing on the ability to prevent urinary tract infections by acidifying the urine and shutting off fim gene expression.

GadE regulates fliC gene transcription and motility in Escherichia coli.

BACKGROUND: Escherichia coli (E. coli) express flagella to ascend human urinary tracts. To survive in the acidic pH of human urine, E. coli uses the glutamate decarboxylase acid response system, which is regulated by the GadE protein. AIM: To determine if growth in an acidic pH environment affected fliC transcription and whether GadE regulated that transcription. METHODS: A fliC-lacZ reporter fusion was created on a single copy number plasmid to assess the effects of acidic pH on fliC transcription. Further, a ΔgadE mutant strain of a uropathogenic E. coli was created and tested for motility compared to the wild-type strain. RESULTS: Escherichia coli cells carrying the fliC-lacZ fusion displayed significantly less fliC transcription when grown in an acidic pH medium compared to when grown in a neutral pH medium. Transcription of fliC fell further when the E. coli was grown in an acidic pH/high osmolarity environment. Since GadE is a critical regulator of one acid response system, fliC transcription was tested in a gadE mutant strain grown under acidic conditions. Expression of fliC was derepressed in the E. coli gadE mutant strain grown under acidic conditions compared to that in wild-type bacteria under the same conditions. Furthermore, a gadE mutation in a uropathogenic E. coli background exhibited significantly greater motility than the wild-type strain following growth in an acidic medium. CONCLUSION: Together, our results suggest that GadE may down-regulate fliC transcription and motility in E. coli grown under acidic conditions.

Ixodes scapularis: Vector to an Increasing Diversity of Human Pathogens in the Upper Midwest.

INTRODUCTION: The black-legged tick, Ixodes scapularis (I scapularis), is now recognized as the deadliest tick vector in the United States. The Upper Midwest, particularly Wisconsin and Minnesota, are endemic to a diversity of tick-transmitted infectious diseases. Although Borrelia burgdorferi, the agent of Lyme disease, still accounts for the majority of diagnosed infections, I scapularis is known to transmit other bacterial, viral, and parasitic agents. OBJECTIVE: To provide an overview of the array of pathogenic microorganisms carried by I scapularis ticks in the Upper Midwest. METHODS: A literature review was conducted to collect and analyze current information about I scapularis lifestyle, transmission, microorganisms carried by the arthropod vector, and the diseases that occur as a result of infections with these microorganisms in the Upper Midwest. RESULTS: Diagnosis of co-infection from tick-borne zoonosis in humans has increased over the last 2 decades. Since I scapularis can transmit multiple pathogens, it is clinically important because different diagnostic testing and treatment strategies may need to be implemented for a patient with I scapularis-borne infection(s). CONCLUSIONS: This review has concentrated on I scapularis-transmitted diseases affecting the Upper Midwest and has explored the ecology of the I scapularis vector and its role in pathogen transmission.

Staphylococcus aureus Toxins: Armaments for a Significant Pathogen.

Staphylococcus species are common inhabitants of humans and other animals [...].

Regulation of the Staphylococcal Superantigen-Like Protein 1 Gene of Community-Associated Methicillin-Resistant Staphylococcus aureus in Murine Abscesses.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) causes substantial skin and soft tissue infections annually in the United States and expresses numerous virulence factors, including a family of toxins known as the staphylococcal superantigen-like (SSL) proteins. Many of the SSL protein structures have been determined and implicated in immune system avoidance, but the full scope that these proteins play in different infection contexts remains unknown and continues to warrant investigation. Analysis of ssl gene regulation may provide valuable information related to the function of these proteins. To determine the transcriptional regulation of the ssl1 gene of CA-MRSA strain MW2, an ssl1 promoter::lux fusion was constructed and transformed into S.aureus strains RN6390 and Newman. Resulting strains were grown in a defined minimal medium (DSM) broth and nutrient-rich brain-heart infusion (BHI) broth and expression was determined by luminescence. Transcription of ssl1 was up-regulated and occurred earlier during growth in DSM broth compared to BHI broth suggesting expression is regulated by nutrient availability. RN6390 and Newman strains containing the ssl1::lux fusion were also used to analyze regulation in vivo using a mouse abscess model of infection. A marked increase in ssl1 transcription occurred early during infection, suggesting SSL1 is important during early stages of infection, perhaps to avoid the immune system.

Virulence factor landscape of a Staphylococcus aureus sequence type 45 strain, MCRF184.

BACKGROUND: We describe the virulence factors of a methicillin-sensitive Staphylococcus aureus sequence type (ST) 45 strain, MCRF184, (spa type t917), that caused severe necrotizing fasciitis in a 72-year-old diabetic male. The genome of MCRF184 possesses three genomic islands: a relatively large type III νSaα with 42 open reading frames (ORFs) that includes superantigen- and lipoprotein-like genes, a truncated νSaß that consists mostly of the enterotoxin gene cluster (egc), and a νSaγ island with 18 ORFs including α-toxin. Additionally, the genome has two phage-related regions: phage φSa3 with three genes of the immune evasion cluster (IEC), and an incomplete phage that is distinct from other S. aureus phages. Finally, the region between orfX and orfY harbors a putative efflux pump, acetyltransferase, regulators, and mobilization genes instead of genes of SCCmec. RESULTS: Virulence factors included phenol soluble modulins (PSMs) α1 through α4 and PSMs ß1 and ß2. Ten ORFs identified in MCRF184 had not been reported in previously sequenced S. aureus strains. CONCLUSION: The dire clinical outcome in the patient and the described virulence factors all suggest that MCRF184, a ST45 strain is a highly virulent strain of S. aureus.

Differential Regulation of Escherichia coli fim Genes following Binding to Mannose Receptors.

Regulation of the uropathogenic Escherichia coli (UPEC) fimB and fimE genes was examined following type 1 pili binding to mannose-coated Sepharose beads. Within 25 min after mannose attachment, fimE expression dropped eightfold, whereas fimB transcription increased about two- to fourfold. Because both fim genes encode site-specific recombinases that affect the position of the fimS element containing the fimA promoter, the positioning of fimS was also examined. The fimS element changed to slightly more Phase-OFF in bacteria mixed with plain beads, whereas UPEC cells interacting with mannose-coated beads had significantly less Phase-OFF orientation of fimS under pH 7 conditions. On the other hand, Phase-OFF oriented fimS increased fourfold when UPEC cells were mixed with plain beads in a pH 5.5 environment. Positioning of fimS was also affected by fimH mutations, demonstrating that the FimH ligand binding to its receptor facilitates the changes. Moreover, enzyme immunoassays showed that UPEC cells had greater type 1 pili expression when mixed with mannose-coated beads versus plain beads. These results indicate that, after type 1 pilus binding to tethered mannose receptors, the physiology of the E. coli cells changes to maintain the expression of type 1 pili even when awash in an acidic environment.

Management of methicillin-resistant Staphylococcus aureus mediated ventilator-associated pneumonia.

Prevention strategies and clinical management of methicillin- resistant Staphylococcus aureus (MRSA) infections in ventilated patients who develop ventilator-associated pneumonia (VAP) are important. Since MRSA are the most frequently isolated bacteria in patients with VAP, and a significant cause of morbidity and mortality in intubated patients, rapid diagnosis and early treatment could reduce mortality. This review will examine preventive steps (i.e. screening ventilated patients for MRSA, decolonization, and hand washing), assessing clinical presentations before the results of culture are obtained to direct empiric treatment, and the appropriate antibiotic therapy upon culture confirmation of MRSA that could help in the management of VAP.

Identification of Staphylococcus aureus Cellular Pathways Affected by the Stilbenoid Lead Drug SK-03-92 Using a Microarray.

The mechanism of action for a new lead stilbene compound coded SK-03-92 with bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA) is unknown. To gain insight into the killing process, transcriptional profiling was performed on SK-03-92 treated vs. untreated S. aureus. Fourteen genes were upregulated and 38 genes downregulated by SK-03-92 treatment. Genes involved in sortase A production, protein metabolism, and transcriptional regulation were upregulated, whereas genes encoding transporters, purine synthesis proteins, and a putative two-component system (SACOL2360 (MW2284) and SACOL2361 (MW2285)) were downregulated by SK-03-92 treatment. Quantitative real-time polymerase chain reaction analyses validated upregulation of srtA and tdk as well as downregulation of the MW2284/MW2285 and purine biosynthesis genes in the drug-treated population. A quantitative real-time polymerase chain reaction analysis of MW2284 and MW2285 mutants compared to wild-type cells demonstrated that the srtA gene was upregulated by both putative two-component regulatory gene mutants compared to the wild-type strain. Using a transcription profiling technique, we have identified several cellular pathways regulated by SK-03-92 treatment, including a putative two-component system that may regulate srtA and other genes that could be tied to the SK-03-92 mechanism of action, biofilm formation, and drug persisters.

Docking into Mycobacterium tuberculosis Thioredoxin Reductase Protein Yields Pyrazolone Lead Molecules for Methicillin-Resistant Staphylococcus aureus.

The thioredoxin/thioredoxin reductase system (Trx/TrxR) is an attractive drug target because of its involvement in a number of important physiological processes, from DNA synthesis to regulating signal transduction. This study describes the finding of pyrazolone compounds that are active against Staphylococcus aureus. Initially, the project was focused on discovering small molecules that may have antibacterial properties targeting the Mycobacterium tuberculosis thioredoxin reductase. This led to the discovery of a pyrazolone scaffold-containing compound series that showed bactericidal capability against S. aureus strains, including drug-resistant clinical isolates. The findings support continued development of the pyrazolone compounds as potential anti-S. aureus antibiotics.

Temporal Regulation of fim Genes in Uropathogenic Escherichia coli during Infection of the Murine Urinary Tract.

Uropathogenic Escherichia coli (UPEC) adhere to cells in the human urinary tract via type 1 pili that undergo phase variation where a 314-bp fimS DNA element flips between Phase-ON and Phase-OFF orientations through two site-specific recombinases, FimB and FimE. Three fim-lux operon transcriptional fusions were created and moved into the clinical UPEC isolate NU149 to determine their temporal regulation in UPEC growing in the urinary tract. Within murine urinary tracts, the UPEC strains demonstrated elevated transcription of fimA and fimB early in the infection, but lower transcription by the fifth day in murine kidneys. In contrast, fimE transcription was much lower than either fimA or fimB early, increased markedly at 24 h after inoculation, and then dropped five days after inoculation. Positioning of fimS was primarily in the Phase-ON position over the time span in UPEC infected bladders, whereas in UPEC infected murine kidneys the Phase-OFF orientation was favored by the fifth day after inoculation. Hemagglutination titers with guinea pig erythrocytes remained constant in UPEC growing in infected murine bladders but fell substantially in UPEC infected kidneys over time. Our results show temporal in vivo regulation of fim gene expression in different environmental niches when UPEC infects the murine urinary tract.

Osmolyte transport in Staphylococcus aureus and the role in pathogenesis.

Osmolyte transport is a pivotal part of bacterial life, particularly in high salt environments. Several low and high affinity osmolyte transport systems have been identified in various bacterial species. A lot of research has centered on characterizing the osmolyte transport systems of Gram-negative bacteria, but less has been done to characterize the same transport systems in Gram-positive bacteria. This review will focus on the previous work that has been done to understand the osmolyte transport systems in the species Staphylococcus aureus and how these transporters may serve dual functions in allowing the bacteria to survive and grow in a variety of environments, including on the surface or within humans or other animals.

Pharmacokinetic/Toxicity Properties of the New Anti-Staphylococcal Lead Compound SK-03-92.

Because of the potential of a new anti-staphylococcal lead compound SK-03-92 as a topical antibiotic, a patch, or an orally active drug, we sought to determine its safety profile and oral bioavailability. SK-03-92 had a high IC50 (125 µg/ml) in vitro against several mammalian cell lines, and mice injected intraperiteonally at the highest dose did not exhibit gross toxicity (e.g. altered gait, ungroomed, significant weight loss). Single dose (100 µg/g) pharmacokinetic (PK) analysis with formulated SK-03-92 showed that peak plasma concentration (1.64 µg/ml) was achieved at 20-30 min. Oral relative bioavailability was 8%, and the drug half-life was 20-30 min, demonstrating that SK-03-92 is likely not a candidate for oral delivery. Five-day and two-week PK analyses demonstrated that SK-03-92 plasma levels were low. Multi-dose analysis showed no gross adverse effects to the mice and a SK-03-92 peak plasma concentration of 2.12 µg/ml with the presence of significant concentrations of breakdown products 15 min after dosing. SK-03-92 appeared to be very safe based on tissue culture and mouse gross toxicity determinations, but the peak plasma concentration suggests that a pro-drug of SK-03-92 or preparation of analogs of SK-03-92 with greater bioavailability and longer half-lives are warranted.

Design and synthesis of novel antimicrobials with activity against Gram-positive bacteria and mycobacterial species, including M. tuberculosis.

The alarming increase in bacterial resistance over the last decade along with a dramatic decrease in new treatments for infections has led to problems in the healthcare industry. Tuberculosis (TB) is caused mainly by Mycobacterium tuberculosis which is responsible for 1.4 million deaths per year. A world-wide threat with HIV co-infected with multi and extensively drug-resistant strains of TB has emerged. In this regard, herein, novel acrylic acid ethyl ester derivatives were synthesized in simple, efficient routes and evaluated as potential agents against several Mycobacterium species. These were synthesized via a stereospecific process for structure activity relationship (SAR) studies. Minimum inhibitory concentration (MIC) assays indicated that esters 12, 13, and 20 exhibited greater in vitro activity against Mycobacterium smegmatis than rifampin, one of the current, first-line anti-mycobacterial chemotherapeutic agents. Based on these studies the acrylic ester 20 has been developed as a potential lead compound which was found to have an MIC value of 0.4 µg/mL against Mycobacterium tuberculosis. The SAR and biological activity of this series is presented; a Michael-acceptor mechanism appears to be important for potent activity of this series of analogs.

OmpR regulation of the uropathogenic Escherichia coli fimB gene in an acidic/high osmolality environment.

Uropathogenic Escherichia coli (UPEC) causes more than 90 % of all human urinary tract infections through type 1 piliated UPEC cells binding to bladder epithelial cells. The FimB and FimE site-specific recombinases orient the fimS element containing the fimA structural gene promoter. Regulation of fimB and fimE depends on environmental pH and osmolality. The EnvZ/OmpR two-component system affects osmoregulation in E. coli. To ascertain if OmpR directly regulated the fimB gene promoters, gel mobility shift and DNase I footprinting experiments were performed using OmpR or phosphorylated OmpR (OmpR-P) mixed with the fimB promoter regions of UPEC strain NU149. Both OmpR-P and OmpR bound weakly to one fimB promoter. Because there was weak binding to one fimB promoter, strain NU149 was grown in different pH and osmolality environments, and total RNAs were extracted from each population and converted to cDNAs. Quantitative reverse-transcriptase PCR showed no differences in ompR transcription among the different growth conditions. Conversely, Western blots showed a significant increase in OmpR protein in UPEC cells grown in a combined low pH/high osmolality environment versus a neutral pH/high osmolality environment. In a high osmolality environment, the ompR mutant expressed more fimB transcripts and Phase-ON positioning of the fimS element as well as higher type 1 pili levels than wild-type cells. Together these results suggest that OmpR may be post-transcriptionally regulated in UPEC cells growing in a low pH/high osmolality environment, which regulates fimB in UPEC.

Antibacterial spirobisnaphthalenes from the North American cup fungus Urnula craterium.

Urnucratins A-C (1-3), which possess an unusual bisnaphthospiroether skeleton with one oxygen bridge and one C-C bridge and represent a new subclass of bisnaphthalenes, were isolated from the North American cup fungus Urnula craterium. Their structures, including absolute configurations, were determined by means of HRMS, NMR, and quantum chemical CD calculations. Urnucratin A (1) was found to be active against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, and Streptococcus pyogenes with MIC values of 2, 1, and 0.5 µg/mL, respectively.

Synthesis and minimum inhibitory concentrations of SK-03-92 against Staphylococcus aureus and other gram-positive bacteria.

Staphylococcus aureus causes hundreds of thousands of infections and thousands of deaths per year in the United States. The emergence of methicillin-resistant S. aureus (MRSA), including community-associated methicillin-resistant S. aureus (CA-MRSA), has added to the problem. As MRSA continue to evolve, they are becoming resistant to more classes of antibiotics. In the past 20 years, only three new antibiotics have been approved for human use (linezolid, daptomycin, and tigecycline), and resistance to these three drugs has already emerged. New antibiotics are needed, and we have developed a promising drug candidate that may be applicable to treating MRSA, among other gram-positive bacterial infections. We have identified a novel synthetic drug, coded SK-03-92, that shows broad in vitro efficacy against a variety of gram-positive bacterial strains that include a number of strains of S. aureus. Besides the activity against gram-positive bacteria, this new drug also exhibits activity against Mycobacterium strains.

Mutational and transcriptional analyses of the Staphylococcus aureus low-affinity proline transporter OpuD during in vitro growth and infection of murine tissues.

Staphylococcus aureus continues to be a major health problem. This species' requirement for proline and proline transport from the extracellular environment is not well understood. Here, we identify a S. aureus low-affinity proline transport gene (opuD) with homology to the OpuD protein of Bacillus subtilis. Mutation of the opuD gene caused a significant decline in proline uptake under low-affinity conditions as compared with wild type, but the opuD mutant strain showed no significant attenuation in a murine abscess model of infection. The S. aureus opuD gene was transcriptionally activated during growth in moderate osmolarity media with high levels of proline or glycine betaine independent of SigB. In murine abscesses, the opuD gene was activated at a later time point, whereas the opuD expression dropped over the course of an 18-h period within murine urinary tracts. Transcriptional regulation of opuD in S. aureus appears to be coordinated within this species when grown in moderate to high NaCl environments, but the level of extracellular proline had a marked effect on expression of this proline transport gene. The differential regulation of proline transport genes in S. aureus may be an adaptation for life in a variety of environments, including survival within the human body.