Lee C.-C.,National Taiwan University |
Yang Y.-C.,National Taiwan University |
Yang Y.-C.,National Taiwan University Hospital |
Goodman S.D.,Center for Microbial Pathogenesis |
And 7 more authors.
Cell and Bioscience | Year: 2015
Background: Deamination of adenine can occur spontaneously under physiological conditions generating the highly mutagenic lesion, hypoxanthine. This process is enhanced by ROS from exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat. Hypoxanthine in DNA can pair with cytosine which results in A:T to G:C transition mutations after DNA replication. In Escherichia coli, deoxyinosine (hypoxanthine deoxyribonucleotide, dI) is removed through an alternative excision repair pathway initiated by endonuclease V. However, the correction of dI in mammalian cells appears more complex and was not fully understood. Results: All four possible dI-containing heteroduplex DNAs, including A-I, C-I, G-I, and T-I were introduced to repair reactions containing extracts from human cells. The repair reaction requires magnesium, dNTPs, and ATP as cofactors. We found G-I was the best substrate followed by T-I, A-I and C-I, respectively. Moreover, judging from the repair requirements and sensitivity to specific polymerase inhibitors, there were overlapping repair activities in processing of dI in DNA. Indeed, a hereditable non-polyposis colorectal cancer cell line (HCT116) demonstrated lower dI repair activity that was partially attributed to lack of mismatch repair. Conclusions: A plasmid-based convenient and non-radioisotopic method was created to study dI repair in human cells. Mutagenic dI lesions processed in vitro can be scored by restriction enzyme cleavage to evaluate the repair. The repair assay described in this study provides a good platform for further investigation of human repair pathways involved in dI processing and their biological significance in mutation prevention. © 2015 Lee et al.
Jurcisek J.A.,Center for Microbial Pathogenesis
Journal of visualized experiments : JoVE | Year: 2011
The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.
A research team led by University of Arkansas chemist Jingyi Chen and University of Arkansas for Medical Sciences microbiologist Mark Smeltzer has developed an alternative therapeutic approach to fighting antibiotic-resistant infections. The novel method uses a targeted, light-activated nanodrug consisting of antibiotic-loaded nanoconstructs, which are nanoscale cages made of gold and coated with polydopamine. The antibiotic is loaded into the polydopamine coating. The gold nanocages convert laser irradiation to heat, resulting in the photothermal effect and simultaneously releasing the antibiotic from the polydopamine coating. “We believe that this approach could facilitate the effective treatment of infections caused by antibiotic-resistant bacteria, including those associated with bacterial biofilms, which are involved in a wide variety of bacterial infections,” says Chen, assistant professor in the Department of Chemistry and Biochemistry in the J. William Fulbright College of Arts and Sciences. Microbial resistance to antibiotics has become a growing public health concern in hospitals and the community at large, so much so that the Infectious Diseases Society of America has designated six bacterial species as “ESKAPE pathogens” — Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. This designation reflects the limited availability of antibiotics that can be used to treat infections caused by these species. “It is also estimated that 80 percent of all bacterial infections involve formation of a biofilm, and all of these infections share the common characteristic of intrinsic resistance to conventional antibiotic therapy,” says Smeltzer, professor in the Department of Microbiology and Immunology at UAMS and director of the Center for Microbial Pathogenesis and Host Inflammatory Responses. “Intrinsic resistance refers to the fact that bacteria within a biofilm exhibit a therapeutically relevant level of resistance to essentially all antibiotics.” Researchers in Smeltzer’s laboratory study the ESKAPE pathogen Staphylococcus aureus. They focus on how the pathogen causes biofilm-associated bone infection and infections associated with orthopaedic implants. But, as Smeltzer explains, there are many other examples in infections — intravenous catheters and vascular grafts, for example — caused by Staphylococcus aureus. The team used Staphylococcus aureus as the proof-of-principle pathogen to demonstrate the potency of their nanodrug. The combination of achieving a photothermal effect and controlled release of antibiotics directly at the site of infection was achieved by laser irradiation at levels within the current safety standard for use in humans. The therapeutic effects of this approach were validated using planktonic bacterial cultures — bacterial cells that are free-floating rather than contained with a biofilm — of both methicillin-sensitive and methicillin-resistant Staphylococcus aureus strains. However, the method was subsequently shown to be effective even in the context of an intrinsically resistant biofilm. “The even better news is that the technology we developed would be readily adaptable to other bacterial pathogens that cause such infections, including the other ESKAPE pathogens,” Smeltzer says. The researchers’ work was recently published in ACS Infectious Diseases, a publication of the American Chemical Society (ACS) and “the first journal to highlight chemistry and its role in the multidisciplinary and collaborative field of infectious disease research.” Participating in the research were first authors Daniel Meeker, an M.D./Ph.D. student in Smeltzer’s lab, and Samir Jenkins, who obtained his doctoral degree in the Chen lab and is now a postdoctoral fellow at UAMS. Other participants included Karen Beenken, senior researcher in Smeltzer’s lab; Allister Loughran at UAMS; Timothy Muldoon, assistant professor of biomedical engineering at the U of A; Amy Powless, doctoral student in biomedical engineering at the U of A; Emily Miller, a U of A undergraduate and Honors College student; Vladimir Zharov, director of the Arkansas Nanomedicine Center at the UAMS Winthrop P. Rockefeller Cancer Institute and professor of otolaryngology, head and neck surgery at UAMS; and Ekaterina Galanzha, associate research professor of otolaryngology, head and neck surgery at UAMS.
Gangaiah D.,Indiana University |
Labandeira-Rey M.,University of Texas Southwestern Medical Center |
Zhang X.,Indiana University |
Fortney K.R.,Indiana University |
And 11 more authors.
mBio | Year: 2014
To adapt to stresses encountered in stationary phase, Gram-negative bacteria utilize the alternative sigma factor RpoS. However, some species lack RpoS; thus, it is unclear how stationary-phase adaptation is regulated in these organisms. Here we defined the growth-phase-dependent transcriptomes of Haemophilus ducreyi, which lacks an RpoS homolog. Compared to mid-log-phase organisms, cells harvested from the stationary phase upregulated genes encoding several virulence determinants and a homolog of hfq. Insertional inactivation of hfq altered the expression of ~16% of the H. ducreyi genes. Importantly, there were a significant overlap and an inverse correlation in the transcript levels of genes differentially expressed in the hfq inactivation mutant relative to its parent and the genes differentially expressed in stationary phase relative to mid-log phase in the parent. Inactivation of hfq downregulated genes in the flp-tad and lspB-lspA2 operons, which encode several virulence determinants. To comply with FDA guidelines for human inoculation experiments, an unmarked hfq deletion mutant was constructed and was fully attenuated for virulence in humans. Inactivation or deletion of hfq downregulated Flp1 and impaired the ability of H. ducreyi to form microcolonies, downregulated DsrA and rendered H. ducreyi serum susceptible, and downregulated LspB and LspA2, which allow H. ducreyi to resist phagocytosis. We propose that, in the absence of an RpoS homolog, Hfq serves as a major contributor of H. ducreyi stationary-phase and virulence gene regulation. The contribution of Hfq to stationary-phase gene regulation may have broad implications for other organisms that lack an RpoS homolog. © 2014 Gangaiah et al.
Gangaiah D.,Indiana University |
Zhang X.,Indiana University |
Fortney K.R.,Indiana University |
Baker B.,Center for Microbial Pathogenesis |
And 4 more authors.
Journal of Bacteriology | Year: 2013
Haemophilus ducreyi causes chancroid, a genital ulcer disease that facilitates the transmission of human immunodeficiency virus type 1. In humans, H. ducreyi is surrounded by phagocytes and must adapt to a hostile environment to survive. To sense and respond to environmental cues, bacteria frequently use two-component signal transduction (2CST) systems. The only obvious 2CST system in H. ducreyi is CpxRA; CpxR is a response regulator, and CpxA is a sensor kinase. Previous studies by Hansen and coworkers showed that CpxR directly represses the expression of dsrA, the lspB-lspA2 operon, and the flp operon, which are required for virulence in humans. They further showed that CpxA functions predominantly as a phosphatase in vitro to maintain the expression of virulence determinants. Since a cpxA mutant is avirulent while a cpxR mutant is fully virulent in humans, CpxA also likely functions predominantly as a phosphatase in vivo. To better understand the role of H. ducreyi CpxRA in controlling virulence determinants, here we defined genes potentially regulated by CpxRA by using RNA-Seq. Activation of CpxR by deletion of cpxA repressed nearly 70% of its targets, including seven established virulence determinants. Inactivation of CpxR by deletion of cpxR differentially regulated few genes and increased the expression of one virulence determinant. We identified a CpxR binding motif that was enriched in downregulated but not upregulated targets. These data reinforce the hypothesis that CpxA phosphatase activity plays a critical role in controlling H. ducreyi virulence in vivo. Characterization of the downregulated genes may offer new insights into pathogenesis. © 2013, American Society for Microbiology.