Multilocus sequence typing (MLST) has been successfully used to differentiate and trace the bacte... more Multilocus sequence typing (MLST) has been successfully used to differentiate and trace the bacterial species and pathogens that cause outbreaks or epidemics of infectious diseases. MLST provides a powerful solution for molecular epidemiological characterization of bacterial strains, including Staphylococcus aureus, by using the sequences of the internal region of seven housekeeping genes. In previous studies, we utilized MLST to analyze the genotypes of S. aureus isolates from pediatric patients with cystic fibrosis and revealed three prevalent ST types, including ST5, ST30, and ST8 in these isolates. In this chapter, we describe a detailed procedure of MLST for genotyping S. aureus.
Biofilm formation on indwelling medical devices represents an exclusive evasion mechanism for man... more Biofilm formation on indwelling medical devices represents an exclusive evasion mechanism for many pathogenic bacteria to establish chronic infections. Staphylococcus aureus is one of the major bacterial pathogens that are able to induce both animal and human infections. The continued emergence of multiple drug-resistant S. aureus, especially methicillin-resistant S. aureus, is problematic due to limited treatment options. Biofilm formation by S. aureus complicates the treatment of methicillin-resistant S. aureus infections. Therefore, elucidating the mechanisms of biofilm formation in this pathogen is important for the development of alternative therapeutic strategies. Various environmental and genetic factors contribute to biofilm formation. In this review, we address the environmental factors and discuss how they affect biofilm formation by S. aureus.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
The emergence of drug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MR... more 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.
Metabolomics is becoming increasingly important in bioscience research as it provides a comprehen... more Metabolomics is becoming increasingly important in bioscience research as it provides a comprehensive analytical platform for a better understanding of the metabolic functions of cells and organisms. Recently, microbial metabolomics has been utilized in diverse research areas, including detection and diagnosis of pathogens, metabolic engineering, and drug discovery. An efficient and reproducible method to measure the intracellular metabolites of a specific microbial organism is a key prerequisite for utilizing metabolome analysis in microbiological research. In this chapter, we describe a workflow focusing on the extraction and quantification of intracellular metabolites of Staphylococcus aureus. Fast quenching with chilled methanol is applied to minimize metabolite leakage, while solvent extraction is used to obtain both polar and nonpolar fractions, which are then analyzed by respective liquid chromatography-mass spectrometry (LC-MS) methods for characterizing and quantifying the intracellular metabolites of S. aureus. This protocol is demonstrated to be an efficient method for analyzing polar and nonpolar intracellular metabolites of S. aureus.
A series of acridone and xanthone-based compounds bearing 1,2-epoxypropyl or 1,2-propanediol subs... more A series of acridone and xanthone-based compounds bearing 1,2-epoxypropyl or 1,2-propanediol substituents were synthesized and evaluated for activity against MRSA and MSSA bacterial strains. The results indicate a correlation exists between the number of epoxide groups and activity, with peak MIC values observed for bis-epoxy derivatives. Both activity and heathy cell toxicity was shown to decrease with the addition of a third epoxy group. The corresponding ring-opened diol analogs were devoid of activity, demonstrating the critical function of the epoxide in mediating antimicrobial activity. The most active compounds were also screened using a regulated antisense RNA expression library. The results show no increase in activity against cells sensitized by down-regulation of the most common drug targets, including DNA gyrase, DNA topoisomerase, tRNA synthetase, and the fatty acid biosynthesis pathway. The compounds are postulated to function as membrane disrupting agents, similar to ...
Rose bengal has been used in the diagnosis of ophthalmic disorders and liver function, and has be... more 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 ...
One of the largest concerns in public health is the continual emergence of multidrug-resistant ba... more 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.
Metabolomics is becoming increasingly important in bioscience research as it provides a comprehen... more Metabolomics is becoming increasingly important in bioscience research as it provides a comprehensive analytical platform for a better understanding of the metabolic functions of cells and organisms. Recently, microbial metabolomics has been utilized in diverse research areas, including detection and diagnosis of pathogens, metabolic engineering, and drug discovery. An efficient and reproducible method to measure the intracellular metabolites of a specific microbial organism is a key prerequisite for utilizing metabolome analysis in microbiological research. In this chapter, we describe a workflow focusing on the extraction and quantification of intracellular metabolites of Staphylococcus aureus. Fast quenching with chilled methanol is applied to minimize metabolite leakage, while solvent extraction is used to obtain both polar and nonpolar fractions, which are then analyzed by respective liquid chromatography-mass spectrometry (LC-MS) methods for characterizing and quantifying the intracellular metabolites of S. aureus. This protocol is demonstrated to be an efficient method for analyzing polar and nonpolar intracellular metabolites of S. aureus.
Discovery of new anti-tuberculosis (TB) drugs is a time-consuming process due to the slow-growing... more 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.
Macrophage polarization is tightly associated with its metabolic reprograming and immune dysfunct... more 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 mitochondria...
Protein–protein interactions among highly conserved and essential proteins can serve as new targe... more Protein–protein interactions among highly conserved and essential proteins can serve as new targets for antibacterial therapies. One protein–protein interaction between two widely conserved and essential bacterial proteins, YeaZ and its paralog, a putative glycoprotease, is being looked into for its antimicrobial drug potential. These two proteins possess tandem functions, including repression of the branched-chain amino acids biosynthesis and induction of a tRNA modification important in enhancing translation fidelity through anticodon–codon base pairing. Heterodimer formation between these two proteins is essential for Staphylococcus aureus, and other bacterial species including Escherichia coli and Salmonella typhimurium. Such YeaZ–glycoprotease interaction could thus be a target for antimicrobial drugs designed for multi-drug-resistant S. aureus. In this review, we discuss the function, structure, and interaction between these two proteins and their orthologs in other bacteria.
The colonization of Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), has... more 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 t...
ABSTRACT The virulence factor α-toxin (hla) is needed by Staphylococcus aureus in order to cause ... more ABSTRACT The virulence factor α-toxin (hla) is needed by Staphylococcus aureus in order to cause infections in both animals and humans. Although the complicated regulation of hla expression has been well studied in human S. aureus isolates, the mechanisms of of hla regulation in bovine S. aureus isolates remain undefined. In this study, we found that many bovine S. aureus isolates, including the RF122 strain, generate dramatic amounts of α-toxin in vitro compared with human clinical S. aureus isolates, including MRSA WCUH29 and MRSA USA300. To elucidate potential regulatory mechanisms, we analyzed the hla promoter regions and identified predominant single nucleotide polymorphisms (SNPs) at positions -376, -483, and -484 from the start codon in α-toxin hyper-producing isolates. Using site-directed mutagenesis and hla promoter-gfp-luxABCDE dual reporter approaches, we demonstrated that the SNPs contribute to the differential control of hla expression among bovine and human S. aureus isolates. Using a DNA affinity assay, gel-shift assays and a null mutant, we identified and revealed that an hla positive regulator, SarZ, contributes to the involvement of the SNPs in mediating hla expression. In addition, we found that the bovine S. aureus isolate RF122 exhibits higher transcription levels of hla positive regulators, including agrA, saeR, arlR and sarZ, but a lower expression level of hla repressor rot compared to the human S. aureus isolate WCUH29. Our results indicate α-toxin hyperproduction in bovine S. aureus is a multifactorial process, influenced at both the genomic and transcriptional levels. Moreover, the identification of predominant SNPs in the hla promoter region may provide a novel method for genotyping the S. aureus isolates.
The hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) strain WCUH29 has bee... more 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).
Frontiers in cellular and infection microbiology, 2017
The SaeRS two-component system plays important roles in regulation of key virulence factors and p... more 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 survi...
Multilocus sequence typing (MLST) has been successfully used to differentiate and trace the bacte... more Multilocus sequence typing (MLST) has been successfully used to differentiate and trace the bacterial species and pathogens that cause outbreaks or epidemics of infectious diseases. MLST provides a powerful solution for molecular epidemiological characterization of bacterial strains, including Staphylococcus aureus, by using the sequences of the internal region of seven housekeeping genes. In previous studies, we utilized MLST to analyze the genotypes of S. aureus isolates from pediatric patients with cystic fibrosis and revealed three prevalent ST types, including ST5, ST30, and ST8 in these isolates. In this chapter, we describe a detailed procedure of MLST for genotyping S. aureus.
Biofilm formation on indwelling medical devices represents an exclusive evasion mechanism for man... more Biofilm formation on indwelling medical devices represents an exclusive evasion mechanism for many pathogenic bacteria to establish chronic infections. Staphylococcus aureus is one of the major bacterial pathogens that are able to induce both animal and human infections. The continued emergence of multiple drug-resistant S. aureus, especially methicillin-resistant S. aureus, is problematic due to limited treatment options. Biofilm formation by S. aureus complicates the treatment of methicillin-resistant S. aureus infections. Therefore, elucidating the mechanisms of biofilm formation in this pathogen is important for the development of alternative therapeutic strategies. Various environmental and genetic factors contribute to biofilm formation. In this review, we address the environmental factors and discuss how they affect biofilm formation by S. aureus.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
The emergence of drug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MR... more 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.
Metabolomics is becoming increasingly important in bioscience research as it provides a comprehen... more Metabolomics is becoming increasingly important in bioscience research as it provides a comprehensive analytical platform for a better understanding of the metabolic functions of cells and organisms. Recently, microbial metabolomics has been utilized in diverse research areas, including detection and diagnosis of pathogens, metabolic engineering, and drug discovery. An efficient and reproducible method to measure the intracellular metabolites of a specific microbial organism is a key prerequisite for utilizing metabolome analysis in microbiological research. In this chapter, we describe a workflow focusing on the extraction and quantification of intracellular metabolites of Staphylococcus aureus. Fast quenching with chilled methanol is applied to minimize metabolite leakage, while solvent extraction is used to obtain both polar and nonpolar fractions, which are then analyzed by respective liquid chromatography-mass spectrometry (LC-MS) methods for characterizing and quantifying the intracellular metabolites of S. aureus. This protocol is demonstrated to be an efficient method for analyzing polar and nonpolar intracellular metabolites of S. aureus.
A series of acridone and xanthone-based compounds bearing 1,2-epoxypropyl or 1,2-propanediol subs... more A series of acridone and xanthone-based compounds bearing 1,2-epoxypropyl or 1,2-propanediol substituents were synthesized and evaluated for activity against MRSA and MSSA bacterial strains. The results indicate a correlation exists between the number of epoxide groups and activity, with peak MIC values observed for bis-epoxy derivatives. Both activity and heathy cell toxicity was shown to decrease with the addition of a third epoxy group. The corresponding ring-opened diol analogs were devoid of activity, demonstrating the critical function of the epoxide in mediating antimicrobial activity. The most active compounds were also screened using a regulated antisense RNA expression library. The results show no increase in activity against cells sensitized by down-regulation of the most common drug targets, including DNA gyrase, DNA topoisomerase, tRNA synthetase, and the fatty acid biosynthesis pathway. The compounds are postulated to function as membrane disrupting agents, similar to ...
Rose bengal has been used in the diagnosis of ophthalmic disorders and liver function, and has be... more 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 ...
One of the largest concerns in public health is the continual emergence of multidrug-resistant ba... more 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.
Metabolomics is becoming increasingly important in bioscience research as it provides a comprehen... more Metabolomics is becoming increasingly important in bioscience research as it provides a comprehensive analytical platform for a better understanding of the metabolic functions of cells and organisms. Recently, microbial metabolomics has been utilized in diverse research areas, including detection and diagnosis of pathogens, metabolic engineering, and drug discovery. An efficient and reproducible method to measure the intracellular metabolites of a specific microbial organism is a key prerequisite for utilizing metabolome analysis in microbiological research. In this chapter, we describe a workflow focusing on the extraction and quantification of intracellular metabolites of Staphylococcus aureus. Fast quenching with chilled methanol is applied to minimize metabolite leakage, while solvent extraction is used to obtain both polar and nonpolar fractions, which are then analyzed by respective liquid chromatography-mass spectrometry (LC-MS) methods for characterizing and quantifying the intracellular metabolites of S. aureus. This protocol is demonstrated to be an efficient method for analyzing polar and nonpolar intracellular metabolites of S. aureus.
Discovery of new anti-tuberculosis (TB) drugs is a time-consuming process due to the slow-growing... more 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.
Macrophage polarization is tightly associated with its metabolic reprograming and immune dysfunct... more 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 mitochondria...
Protein–protein interactions among highly conserved and essential proteins can serve as new targe... more Protein–protein interactions among highly conserved and essential proteins can serve as new targets for antibacterial therapies. One protein–protein interaction between two widely conserved and essential bacterial proteins, YeaZ and its paralog, a putative glycoprotease, is being looked into for its antimicrobial drug potential. These two proteins possess tandem functions, including repression of the branched-chain amino acids biosynthesis and induction of a tRNA modification important in enhancing translation fidelity through anticodon–codon base pairing. Heterodimer formation between these two proteins is essential for Staphylococcus aureus, and other bacterial species including Escherichia coli and Salmonella typhimurium. Such YeaZ–glycoprotease interaction could thus be a target for antimicrobial drugs designed for multi-drug-resistant S. aureus. In this review, we discuss the function, structure, and interaction between these two proteins and their orthologs in other bacteria.
The colonization of Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), has... more 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 t...
ABSTRACT The virulence factor α-toxin (hla) is needed by Staphylococcus aureus in order to cause ... more ABSTRACT The virulence factor α-toxin (hla) is needed by Staphylococcus aureus in order to cause infections in both animals and humans. Although the complicated regulation of hla expression has been well studied in human S. aureus isolates, the mechanisms of of hla regulation in bovine S. aureus isolates remain undefined. In this study, we found that many bovine S. aureus isolates, including the RF122 strain, generate dramatic amounts of α-toxin in vitro compared with human clinical S. aureus isolates, including MRSA WCUH29 and MRSA USA300. To elucidate potential regulatory mechanisms, we analyzed the hla promoter regions and identified predominant single nucleotide polymorphisms (SNPs) at positions -376, -483, and -484 from the start codon in α-toxin hyper-producing isolates. Using site-directed mutagenesis and hla promoter-gfp-luxABCDE dual reporter approaches, we demonstrated that the SNPs contribute to the differential control of hla expression among bovine and human S. aureus isolates. Using a DNA affinity assay, gel-shift assays and a null mutant, we identified and revealed that an hla positive regulator, SarZ, contributes to the involvement of the SNPs in mediating hla expression. In addition, we found that the bovine S. aureus isolate RF122 exhibits higher transcription levels of hla positive regulators, including agrA, saeR, arlR and sarZ, but a lower expression level of hla repressor rot compared to the human S. aureus isolate WCUH29. Our results indicate α-toxin hyperproduction in bovine S. aureus is a multifactorial process, influenced at both the genomic and transcriptional levels. Moreover, the identification of predominant SNPs in the hla promoter region may provide a novel method for genotyping the S. aureus isolates.
The hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) strain WCUH29 has bee... more 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).
Frontiers in cellular and infection microbiology, 2017
The SaeRS two-component system plays important roles in regulation of key virulence factors and p... more 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 survi...
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