Antibacterial Activity of Pharmaceutical-Grade Rose Bengal: An Application of a Synthetic Dye in Antibacterial Therapies.

Rose bengal has been used in the diagnosis of ophthalmic disorders and liver function, and has been studied for the treatment of solid tumor cancers. To date, the antibacterial activity of rose bengal has been sporadically reported; however, these data have been generated with a commercial grade of rose bengal, which contains major uncontrolled impurities generated by the manufacturing process (80-95% dye content). A high-purity form of rose bengal formulation (HP-RBf, >99.5% dye content) kills a battery of Gram-positive bacteria, including drug-resistant strains at low concentrations (0.01-3.13 µg/mL) under fluorescent, LED, and natural light in a few minutes. Significantly, HP-RBf effectively eradicates Gram-positive bacterial biofilms. The frequency that Gram-positive bacteria spontaneously developed resistance to HP-RB is extremely low (less than 1 × 10-13). Toxicity data obtained through our research programs indicate that HP-RB is feasible as an anti-infective drug for the treatment of skin and soft tissue infections (SSTIs) involving multidrug-resistant (MDR) microbial invasion of the skin, and for eradicating biofilms. This article summarizes the antibacterial activity of pharmaceutical-grade rose bengal, HP-RB, against Gram-positive bacteria, its cytotoxicity against skin cells under illumination conditions, and mechanistic insights into rose bengal's bactericidal activity under dark conditions.

SATB1 3'-UTR and lncRNA-UCA1 competitively bind to miR-495-3p and together regulate the proliferation and invasion of gastric cancer.

High expression of special AT-rich-binding protein 1 (SATB1) correlates with the advanced TNM stage and short overall and recurrence-free survival of gastric cancer (GC). A bioinformatic analysis revealed that SATB1 3'-untranslated region (3'-UTR) and long noncoding RNA UCA1 (lncRNA-UCA1) might competitively bind to microRNA-495-3p (miR-495-3p). Interestingly, lncRNA-UCA1 is also an important contributor to GC. The current study aimed to demonstrate the potential interaction among SATB1/miR-495-3p/lncRNA-UCA1 network and their effects on GC proliferation and invasion. The expression in GC and paracancerous normal tissues were assessed using real-time polymerase chain reaction and Western blot analysis. Luciferase reporter, RNA pull-down, and transfection assays were performed to determine the interaction among SATB1/miR-495-3p/lncRNA-UCA1 network in GC cells. GC proliferation and invasion were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, transwell invasion, and colony formation assays. Results showed higher expression of SATB1 and lncRNA-UCA1 but lower miR-495-3p expression in GC than in the normal tissues. In luciferase reporter assay, miR-495-3p bound to three seed sequences in SATB1 3'-UTR but only one in lncRNA-UCA1. SATB1 knockdown increased the combination of miR-495-3p with lncRNA-UCA1 but decreased lncRNA-UCA1 expression. Decreased lncRNA-UCA1 was also observed with the mimics increased miR-495-3p. These data suggested that SATB1 3'-UTR functions as a competing endogenous RNA of miR-495-3p and positively regulates lncRNA-UCA1. LncRNA-UCA1 knockdown only decreased SATB1 expression in MKN-45 cells but not in BGC-823 cells, which suggested that the regulatory effect of lncRNA-UCA1 on SATB1 by sponging miR-495-3p is cell-dependent. This study further identified that SATB1/miR-495-3p/lncRNA-UCA1 network is implicated in GC proliferation and invasion. The current study firstly revealed that SATB1 interacts with miR-495-3p/lncRNA-UCA1 network, whereby enhancing GC proliferation and invasion.

The Staphylococcus aureus AirSR Two-Component System Mediates Reactive Oxygen Species Resistance via Transcriptional Regulation of Staphyloxanthin Production.

Staphylococcus aureus is an important opportunistic pathogen and is the etiological agent of many hospital- and community-acquired infections. The golden pigment, staphyloxanthin, of S. aureus colonies distinguishes it from other staphylococci and related Gram-positive cocci. Staphyloxanthin is the product of a series of biosynthetic steps that produce a unique membrane-embedded C30 golden carotenoid and is an important antioxidant. We observed that a strain with an inducible airR overexpression cassette had noticeably increased staphyloxanthin production compared to the wild-type strain under aerobic culturing conditions. Further analysis revealed that depletion or overproduction of the AirR response regulator resulted in a corresponding decrease or increase in staphyloxanthin production and susceptibility to killing by hydrogen peroxide, respectively. Furthermore, the genetic elimination of staphyloxanthin during AirR overproduction abolished the protective phenotype of increased staphyloxanthin production in a whole-blood survival assay. Promoter reporter and gel shift assays determined that the AirR response regulator is a direct positive regulator of the staphyloxanthin-biosynthetic operon, crtOPQMN, but is epistatic to alternative sigma factor B. Taken together, these data indicate that AirSR positively regulates the staphyloxanthin-biosynthetic operon crtOPQMN, promoting survival of S. aureus in the presence of oxidants.

Characterization of Staphylococcus aureus isolates from pediatric patients with cystic fibrosis.

Staphylococcus aureus is one of the major respiratory pathogens associated with cystic fibrosis (CF) patients. In this study, we collected sputum and isolated fifty S. aureus isolates from CF patients with the median age of 9.5 years old. Then we determined the profiles of these isolates by antibiotic susceptibility testing, examining their cytotoxicity and ability to internalize into an epithelial cell line (A549), as well as multiple loci sequencing typing. Predominant CF S. aureus isolates were resistant to penicillin; however, these isolates were sensitive to various antibiotics, such as vancomycin and minocycline. Different CF S. aureus isolates showed distinct cytotoxic activities, and 90 % of CF S. aureus isolates possessed the enterotoxin genes, sea and hlg. Moreover, we found that multiple different CF S. aureus isolates appeared to have the distinct capacity of invading A549 cells. ST5 (14 %), ST30 (14 %), and ST8 (10 %) were prevalent ST types in these isolates. Further analysis revealed that ST5 and ST30 isolates were less toxic than ST8 and ST15 isolates, and that the ST5, ST15, ST59, and ST87 types of CF S. aureus were less capable of invading A549 cells. Our results suggest that the ST typing method may be useful in predicting cytotoxicity and the invading capacity of S. aureus isolates from patients with CF.

New Insights into the Biological Functions of Essential TsaB/YeaZ Protein in Staphylococcus aureus.

TsaB/YeaZ represents a promising target for novel antibacterial agents due to its indispensable role in bacterial survival, high conservation within bacterial species, and absence of eukaryotic homologs. Previous studies have elucidated the role of the essential staphylococcal protein, TsaB/YeaZ, in binding DNA to mediate the transcription of the ilv-leu operon, responsible for encoding key enzymes involved in the biosynthesis of branched-chain amino acids-namely isoleucine, leucine, and valine (ILV). However, the regulation of ILV biosynthesis does not account for the essentiality of TsaB/YeaZ for bacterial growth. In this study, we investigated the impact of TsaB/YeaZ depletion on bacterial morphology and gene expression profiles using electron microscopy and deep transcriptomic analysis, respectively. Our results revealed significant alterations in bacterial size and surface smoothness upon TsaB/YeaZ depletion. Furthermore, we pinpointed specific genes and enriched biological pathways significantly affected by TsaB/YeaZ during the early and middle exponential phases and early stationary phases of growth. Crucially, our research uncovered a regulatory role for TsaB/YeaZ in bacterial autolysis. These discoveries offer fresh insights into the multifaceted biological functions of TsaB/YeaZ within S. aureus.

Targeting multidrug resistant Staphylococcus infections with bacterial histidine kinase inhibitors.

The emergence of drug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), which are not susceptible to current antibiotics has necessitated the development of novel approaches and targets to tackle this growing challenge. Bacterial two-component systems (TCSs) play a central role in the adaptative response of bacteria to their ever-changing environment. They are linked to antibiotic resistance and bacterial virulence making the proteins of the TCSs, histidine kinases and response regulators, attractive for the development of novel antibacterial drugs. Here, we developed a suite of maleimide-based compounds that we evaluated against a model histidine kinase, HK853, in vitro and in silico. The most potent leads were then assessed for their ability to decrease the pathogenicity and virulence of MRSA, resulting in the identification of a molecule that decreased the lesion size caused by a methicillin-resistant S. aureus skin infection by 65% in a murine model.

Identification and Validation of a Novel Antibacterial Compound MZ-01 against Methicillin-Resistant Staphylococcus aureus.

The discovery of new classes of antibiotics is slow, and it is being greatly outpaced by the development of bacterial resistance. This disparity places us in an increasingly vulnerable position because we are running out of safe and effective therapeutic options to treat antibiotic-resistant infections. This is exemplified by the emergence and persistence of hospital-acquired and community-associated methicillin-resistant S. aureus (MRSA), which has markedly narrowed our options for treating life-threatening staph infections. Thus, there is an urgent need to develop novel, potent, preventive, and therapeutic agents. In our current study, we performed a whole-cell screening assay of synthetic libraries for antibacterial activity and identified a novel molecule, MZ-01. MZ-01 exhibited potent bactericidal activity against Gram-positive bacterial pathogens, including MRSA, Streptococcus pyogenes, and Streptococcus pneumoniae, at low concentrations. MZ-01 killed and lysed both the late exponential phase of an S. aureus population and bacteria inside mammalian cells. Furthermore, MZ-01 exhibited low cytotoxicity. These results indicate that MZ-01 is a promising scaffold to guide the development of novel, potent antibacterial agents against multidrug-resistant Gram-positive bacterial pathogens such as MRSA.

The essential two-component system YhcSR is involved in regulation of the nitrate respiratory pathway of Staphylococcus aureus.

Our previous studies revealed that a novel two-component signal transduction system, YhcSR, is essential for the survival of Staphylococcus aureus; however, the biological function of YhcSR remains unknown. In this study, we demonstrated that YhcSR plays an important role in the modulation of the nitrate respiratory pathway under anaerobic conditions. Specifically, we determined that nitrate induces yhcS transcription in the early log phase of growth under anaerobic conditions and that the downregulation of yhcSR expression eliminates the stimulatory effect of nitrate on bacterial growth. Using semiquantitative real-time reverse transcription-PCR (qPCR) and promoter-lux reporter fusions, we established that YhcSR positively modulates the transcription of the narG operon, which is involved in the nitrate respiratory pathway. Our gel shift assays revealed that YhcR binds to the promoter regions of narG and nreABC. Collectively, the above data indicate that the yhcSR system directly regulates the expression of both narG and nreABC operons, which in turn positively modulate the nitrate respiratory pathway of S. aureus under anaerobic conditions. These results provide a new insight into the biological functions of the essential two-component YhcSR system.

Identification of cbiO Gene Critical for Biofilm Formation by MRSA CFSa36 Strain Isolated from Pediatric Patient with Cystic Fibrosis.

The colonization of Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), has a detrimental effect on the respiratory care of pediatric patients with cystic fibrosis (CF). In addition to being resistant to multiple antibiotics, S. aureus also has the ability to form biofilms, which makes the infection more difficult to treat and eradicate. In this study, we examined the ability of S. aureus strains isolated from pediatric patients with CF to form biofilms. We screened a transposon mutant library of MRSA and identified a putative cobalt transporter ATP binding domain (cbiO) that is required for biofilm formation. We discovered that deleting cbiO creating a cbiO null mutant in CFSa36 (an MRSA strain isolated from a patient with cystic fibrosis) significantly hinders the ability of CFSa36 to form biofilm. The complementation of cbiO restored the ability of the cbiO deletion mutant to generate biofilm. Interestingly, we revealed that incorporating extra copper ions to the chemically defined medium (CDM) complemented the function of cbiO for biofilm formation in a dose-dependent manner, while the addition of extra iron ions in CDM enhanced the effect of cbiO null mutation on biofilm formation. In addition, neither the addition of certain extra amounts of copper ions nor iron ions in CDM had an impact on bacterial growth. Taken together, our findings suggest that cbiO mediates biofilm formation by affecting the transportation of copper ions in the MRSA CFSa36 strain. This study provides new insights into the molecular basis of biofilm formation by S. aureus.

Contribution of Coagulase and Its Regulator SaeRS to Lethality of CA-MRSA 923 Bacteremia.

Coagulase is a critical factor for distinguishing Staphylococcus aureus and coagulase-negative Staphylococcus. Our previous studies demonstrated that the null mutation of coagulase (coa) or its direct regulator, SaeRS, significantly enhanced the ability of S. aureus (CA-MRSA 923) to survive in human blood in vitro. This led us to further investigate the role of coagulase and its direct regulator, SaeRS, in the pathogenicity of CA-MRSA 923 in bacteremia during infection. In this study, we found that the null mutation of coa significantly decreased the mortality of CA-MRSA 923; moreover, the single null mutation of saeRS and the double deletion of coa/saeRS abolished the virulence of CA-MRSA 923. Moreover, the mice infected with either the saeRS knockout or the coa/saeRS double knockout mutant exhibited fewer histological lesions and less neutrophils infiltration in the infected kidneys compared to those infected with the coa knockout mutant or their parental control. Furthermore, we examined the impact of coa and saeRS on bacterial survival in vitro. The null mutation of coa had no impact on bacterial survival in mice blood, whereas the deletion mutation of saeRS or coa/saeRS significantly enhanced bacterial survival in mice blood. These data indicate that SaeRS plays a key role in the lethality of CA-MRSA 923 bacteremia, and that coagulase is one of the important virulence factors that is regulated by SaeRS and contributes to the pathogenicity of CA-MRSA 923.

Identification of Target Genes Mediated by Two-Component Regulators of Staphylococcus aureus Using RNA-seq Technology.

Transcriptomics enables us to elucidate comprehensive gene expression profiles in given experimental conditions. Global regulators, which include transcriptional regulators and two-component regulatory systems, have evolved in a variety of bacterial systems. They play important roles in bacterial fitness and pathogenesis by regulating target gene expression. Advanced next-generation RNA sequencing technology (RNA-seq) provides a powerful and effective tool to analyze the transcriptome of bacterial cells. In this chapter, we provide a detailed procedure for the investigation of gene expression profiles and identification of target genes, regulons, and/or pathways that are mediated by a regulator. This procedure is done using RNA-seq analysis, which involves RNA purification, mRNA enrichment, decontamination, RNA-seq data analysis, and quantitative real-time reverse transcription PCR.

Determining Impact of Growth Phases on Capacity of Staphylococcus aureus to Adhere to and Invade Host Cells.

One of the largest concerns in public health is the continual emergence of multidrug-resistant bacterial pathogens. The resistance of bacterial pathogens to specific drugs presents a significant problem because it severely limits treatment options. Staphylococcus aureus is a particularly problematic pathogen that is prevalent in human and animal populations. Data on this bacterium have shown that S. aureus is capable of invading different types of host cells, suggesting that multiple mechanisms are behind its ability to thwart a host immune system and evade the toxicity of some antibiotics. S. aureus produces a myriad of cell wall-associated molecules, such as fibronectin-binding proteins, which assist in the adhesion and invasion of the bacterial cell to a host cell. Understanding the expression of these cell wall-associated molecules at different growth phases will improve general knowledge on how this bacterium can adhere to and invade a host. In our previous work, we found that different types of human MRSA isolates possess different abilities to adhere to and invade epithelial cells. In a recent study we conducted, it was found that S. aureus taken from the exponential phase of growth, when compared to S. aureus taken from the stationary phase, had a noticeable higher ability to invade host cells.

Application of Mycobacterium smegmatis as a surrogate to evaluate drug leads against Mycobacterium tuberculosis.

Discovery of new anti-tuberculosis (TB) drugs is a time-consuming process due to the slow-growing nature of Mycobacterium tuberculosis (Mtb). A requirement of biosafety level 3 (BSL-3) facility for performing research associated with Mtb is another limitation for the development of TB drug discovery. In our screening of BSL-1 Mycobacterium spp. against a battery of TB drugs, M. smegmatis (ATCC607) exhibits good agreement with its drug susceptibility against the TB drugs under a low-nutrient culture medium (0.5% Tween 80 in Middlebrook 7H9 broth). M. smegmatis (ATCC607) enters its dormant form in 14 days under a nutrient-deficient condition (a PBS buffer), and shows resistance to a majority of TB drugs, but shows susceptibility to amikacin, capreomycin, ethambutol, and rifampicin (with high concentrations) whose activities against non-replicating (or dormant) Mtb were previously validated.

Development of shuttle vectors for evaluation of essential regulator regulated gene expression in Staphylococcus aureus.

We describe the construction of a series of shuttle vectors for Staphylococcus aureus. In order to determine transcriptional regulation by essential regulators, we constructed promoterless luxABCDE reporter system using a TetR-regulated antisense RNA expression vector, pJYJ909, which is composed of S. aureus plasmid pE194, the Gram(-) plasmid pUC18, a TetR regulatory cassette, and Pxyl/teto-driven yhcS antisense expression construct. The reformed shuttle vector was utilized to construct an opuCA promoter-luxABCDE fusion and simultaneously examine transcriptional regulation by measuring bioluminescence intensity during down-regulating yhcSR. In addition, we utilized the same plasmid, pJYJ909, and constructed a Pspac-driven constant expression system, which allows us to determine the complementary effect of overexpression of opuCA operon modulated by yhcSR. These plasmids provide important tools for elucidating regulatory mechanisms for genes that are essential for bacterial growth in S. aureus.

Complete Genome Sequence of Hospital-Acquired Methicillin-Resistant Staphylococcus aureus Strain WCUH29.

The hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) strain WCUH29 has been intensively and widely used as a model system for identification and evaluation of novel antibacterial targets and pathogenicity. In this announcement, we report the complete genome sequence of HA-MRSA WCUH29 (NCIMB 40771).

Down-regulation of guanylate binding protein 1 causes mitochondrial dysfunction and cellular senescence in macrophages.

Macrophage polarization is tightly associated with its metabolic reprograming and immune dysfunction. However, the intracellular molecules/pathways that connect these alterations in inflammatory macrophages remain largely unidentified. Herein, we explored the role of guanylate binding protein 1 (Gbp1), an intracellular anti-microbial protein, in regulating polarization, metabolic reprogramming, and cellular aging of macrophages. We showed that Gbp1 expression in inguinal white adipose tissue is significantly decreased in high-fat diet -fed and aged mice. Gbp1 expression is significantly induced by IFNγ and LPS in macrophages but not adipocytes. Downregulation of Gbp1 expression causes macrophage polarization towards a pro-inflammatory phenotype. Gbp1 knockdown (Kd) macrophages have impaired mitochondrial respiratory function, which is further supported by down-regulation of genes encoding electron transport chain components and genes involved in fatty acid oxidation and mitochondrial function. Moreover, we observed Gbp1 is localized in both cytosol and mitochondrial fraction, and Gbp1 Kd macrophages display decreased mitophagy activity. More interestingly, Gbp1 Kd macrophages undergo senescence as evidenced by increased activation of AMPK-p53 pathway and positive staining of ß-galactosidase. These observations suggest that Gbp1 may play an important role in protecting against mitochondrial dysfunction and preserving immune function of macrophages during inflammatory stress and aging.

The SaeRS Two-Component System Controls Survival of Staphylococcus aureus in Human Blood through Regulation of Coagulase.

The SaeRS two-component system plays important roles in regulation of key virulence factors and pathogenicity. In this study, however, we found that the deletion mutation of saeRS enhanced bacterial survival in human blood, whereas complementation of the mutant with SaeRS returned survival to wild-type levels. Moreover, these phenomena were observed in different MRSA genetic background isolates, including HA-MRSA WCUH29, CA-MRSA 923, and MW2. To elucidate which gene(s) regulated by SaeRS contribute to the effect, we conducted a series of complementation studies with selected known SaeRS target genes in trans. We found coagulase complementation abolished the enhanced survival of the SaeRS mutant in human blood. The coa and saeRS deletion mutants exhibited a similar survival phenotype in blood. Intriguingly, heterologous expression of coagulase decreased survival of S. epidermidis in human blood. Further, the addition of recombinant coagulase to blood significantly decreased the survival of S. aureus. Further, analysis revealed staphylococcal resistance to killing by hydrogen peroxide was partially dependent on the presence or absence of coagulase. Furthermore, complementation with coagulase, but not SaeRS, returned saeRS/coa double mutant survival in blood to wild-type levels. These data indicate SaeRS modulates bacterial survival in blood in coagulase-dependent manner. Our results provide new insights into the role of staphylococcal SaeRS and coagulase on bacterial survival in human blood.

A New Combination of a Pleuromutilin Derivative and Doxycycline for Treatment of Multidrug-Resistant Acinetobacter baumannii.

Multidrug-resistant (MDR) Acinetobacter baumannii is one of the most difficult Gram-negative bacteria to treat and eradicate. In a cell-based screening of pleuromutilin derivatives against a drug sensitive A. baumannii strain, new molecules (2-4) exhibit bacteriostatic activity with 3.13 µg/mL concentration and 1 shows bactericidal activity with an MBC of 6.25 µg/mL. The pleuromutilin derivative 1 displays strong synergistic effects with doxycycline in a wide range of concentrations. A 35/1 ratio of 1 and doxycycline (1-Dox 35/1) kills drug susceptible A. baumannii with the MBC of 2.0 µg/mL and an MDR A. baumannii with the MBC of 3.13 µg/mL. In vitro anti-Acinetobacter activity of 1-Dox 35/1 is superior to that of clinical drugs such as tobramycin, tigecycline, and colistin. The efficacy of 1-Dox 35/1 is evaluated in a mouse septicemia model; treatment of the infected C57BL/6 mice with 1-Dox 35/1 protects from lethal infection of A. baumannii with an ED50 value of <2.0 mg/kg.

Identification of an essential glycoprotease in Staphylococcus aureus.

The emergence of multi-drug resistant bacterial pathogens is generating enormous public health concern, and highlights an urgent need for new, alternative agents for treating multi-drug-resistant pathogens. The gene products essential for bacterial growth in vitro and survival during infection constitute an initial set of protein targets for the development of antibacterial agents. In this study, we employed regulated gene expression approaches and demonstrated that a putative glycoprotease (Gcp) is required for staphylococcal growth in the culture. We found that Staphylococcus aureus becomes more sensitive to the Zn(2+) ion under the downregulation of Gcp expression in vitro. Bioinformatic analyses demonstrated that Gcp is conserved in many Gram-positive pathogens and exists in a variety of Gram-negative pathogens. Our results indicate that Gcp is a potential novel target for the development of antimicrobials against S. aureus infection.

Determination of essentiality and regulatory function of staphylococcal YeaZ in branched-chain amino acid biosynthesis.

The staphylococcal YeaZ is highly conserved in prokaryotic cells and critical for growth of many bacterial pathogens. However, the essentiality for Staphylococcus aureus growth and the biological function of YeaZ behind its essentiality remain undefined. In this study, we created and characterized a defined Pspac-regulated yeaZ expression mutant in S. aureus and demonstrated the indispensability of YeaZ for S. aureus growth. Moreover, we conducted complementation studies, not only confirmed the requirement of YeaZ for S. aureus growth, but also revealed a similarity of essential function between staphylococcal YeaZ and its E. coli homolog. On the other hand, we explored the biological functions of YeaZ and found that YeaZ is involved in the regulation of the transcription of ilv-leu operon that encodes key enzymes responsible for the biosynthesis of the branched-chain amino acids, including isoleucine, leucine, and valine (ILV). qPCR analysis showed that the 6-fold downregulation of YeaZ dramatically elevated approximately 17- to 289-fold RNA levels of ilvD, leuA and ilvA. We further confirmed the transcriptional regulation of the ilv-leu operon by YeaZ using an ilv-promoter-lux reporter system and gel-shift assays and revealed that YeaZ is able to bind the promoter region of ilv. Furthermore, we established that the regulation of ILV biosynthesis isn't associated with YeaZ's essentiality, as the deletion of the ilv-leu operon did not affect the requirement of YeaZ for growth in culture. Our results demonstrate the essentiality of YeaZ for S. aureus growth and suggest that the staphylococcal YeaZ possesses regulatory function.