Faculty Bibliography
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339
Repair of global regulators in Staphylococcus aureus 8325 and comparative analysis with other clinical isolates
The pathogenicity of Staphylococcus aureus strains varies tremendously (as seen with animals). It is largely dependent on global regulators, which control the production of toxins, virulence, and fitness factors. Despite the vast knowledge of staphylococcal molecular genetics, there is still widespread dispute over what factors must come together to make a strain highly virulent. S. aureus NCTC8325 (RN1 and derivatives) is a widely used model strain for which an incomparable wealth of knowledge has accumulated in the almost 50 years since its isolation. Although RN1 has functional agr, sarA, and sae global regulators, it is defective in two regulatory genes, rsbU (a positive activator of SigB) and tcaR (an activator of protein A transcription), and is therefore considered by many to be a poor model for studies of regulation and virulence. Here, we repaired these genes and compared the resulting RN1 derivatives with other widely used strains, Newman, USA300, UAMS-1, and COL, plus the parental RN1, with respect to growth, extracellular protein pattern, hemolytic activity, protein A production, pigmentation, biofilm formation, and mouse lethality. The tcaR-repaired strain, showed little alteration in these properties. However, the rsbU-repaired strain was profoundly altered. Hemolytic activity was largely decreased, the exoprotein pattern became much more similar to that of typical wild-type (wt) S. aureus, and there was a surprising increase in mouse lethality. We note that each of the strains tested has a mutational alteration in one or more other regulatory functions, and we conclude that the repaired RN1 is a good model strain for studies of staphylococcal regulation and pathobiology; although strain Newman has been used extensively for such studies in recent years, it has a missense mutation in saeS, the histidine kinase component of the sae signaling module, which profoundly alters its regulatory phenotype. If this mutation were repaired, Newman would be considerably improved as a model strain.
PMCID:2876537
PMID: 20212089
ISSN: 1098-5522
CID: 2456942
Climate change and the integrity of science [Letter]
PMCID:5125622
PMID: 20448167
ISSN: 0036-8075
CID: 210722
Symmetric signalling within asymmetric dimers of the Staphylococcus aureus receptor histidine kinase AgrC
Virulence in Staphylococcus aureus is largely under control of the accessory gene regulator (agr) quorum-sensing system. The AgrC receptor histidine kinase detects its autoinducing peptide (AIP) ligand and generates an intracellular signal resulting in secretion of virulence factors. Although agr is a well-studied quorum-sensing system, little is known about the mechanism of AgrC activation. By co-immunoprecipitation analysis and intermolecular complementation of receptor mutants, we showed that AgrC forms ligand-independent dimers that undergo trans-autophosphorylation upon interaction with AIP. Remarkably, addition of specific AIPs to AgrC mutant dimers with only one functional sensor domain caused symmetric activation of either kinase domain despite the sensor asymmetry. Furthermore, mutant dimers involving one constitutive protomer demonstrated ligand-independent activity, irrespective of which protomer was kinase deficient. These results demonstrate that signalling through either individual AgrC protomer causes symmetric activation of both kinase domains. We suggest that such signalling across the dimer interface may be an important mechanism for dimeric quorum-sensing receptors to rapidly elicit a response upon signal detection
PMCID:3913215
PMID: 19708918
ISSN: 1365-2958
CID: 106013
Specificity of staphylococcal phage and SaPI DNA packaging as revealed by integrase and terminase mutations
SaPI1 and SaPIbov1 are chromosomal pathogenicity islands in Staphylococcus aureus that carry tst and other superantigen genes. They are induced to excise and replicate by certain phages, are efficiently encapsidated in SaPI-specific small particles composed of phage virion proteins and are transferred at very high frequencies. In this study, we have analysed three SaPI genes that are important for the phage-SaPI interaction, int (integrase) terS (phage terminase small subunit homologue) and pif (phage interference function). SaPI1 int is required for SaPI excision, replication and packaging in a donor strain, and is required for integration in a recipient. A SaPI1 int mutant, following phage induction, produces small SaPI-specific capsids which are filled with partial phage genomes. SaPIbov1 DNA is efficiently packaged into full-sized phage heads as well as into SaPI-specific small ones, whereas SaPI1 DNA is found almost exclusively in the small capsids. TerS, however, determines DNA packaging specificity but not the choice of large versus small capsids. This choice is influenced by SaPIbov1 gene 12, which prevents phage DNA packaging into small capsids, and which is also primarily responsible for interference by SaPIbov1 with phage reproduction
PMCID:3885990
PMID: 19347993
ISSN: 1365-2958
CID: 97845
Proceedings of the National Academy of Sciences of the United States of America (PNAS). 2009:106(4):1234-8.DOI: 10.1073/pnas.0809600106
Killing niche competitors by remote-control bacteriophage induction
A surprising example of interspecies competition is the production by certain bacteria of hydrogen peroxide at concentrations that are lethal for others. A case in point is the displacement of Staphylococcus aureus by Streptococcus pneumoniae in the nasopharynx, which is of considerable clinical significance. How it is accomplished, however, has been a great mystery, because H(2)O(2) is a very well known disinfectant whose lethality is largely due to the production of hyperoxides through the abiological Fenton reaction. In this report, we have solved the mystery by showing that H(2)O(2) at the concentrations typically produced by pneumococci kills lysogenic but not nonlysogenic staphylococci by inducing the SOS response. The SOS response, a stress response to DNA damage, not only invokes DNA repair mechanisms but also induces resident prophages, and the resulting lysis is responsible for H(2)O(2) lethality. Because the vast majority of S. aureus strains are lysogenic, the production of H(2)O(2) is a very widely effective antistaphylococcal strategy. Pneumococci, however, which are also commonly lysogenic and undergo SOS induction in response to DNA-damaging agents such as mitomycin C, are not SOS-induced on exposure to H(2)O(2). This is apparently because they are resistant to the DNA-damaging effects of the Fenton reaction. The production of an SOS-inducing signal to activate prophages in neighboring organisms is thus a rather unique competitive strategy, which we suggest may be in widespread use for bacterial interference. However, this strategy has as a by-product the release of active phage, which can potentially spread mobile genetic elements carrying virulence genes
PMCID:2633583
PMID: 19141630
ISSN: 1091-6490
CID: 106012
Proceedings of the National Academy of Sciences of the United States of America (PNAS). 2009:106(4):1216-21.DOI: 10.1073/pnas.0807760106
agr receptor mutants reveal distinct modes of inhibition by staphylococcal autoinducing peptides
Through the agr quorum-sensing system, staphylococci secrete unique autoinducing peptides (AIPs) and detect their concentration via the AgrC transmembrane receptor, coordinating local bacterial population density with global changes in gene expression. Unique AIP and AgrC variants exist within and between species, and although autologous interactions lead to agr activation, heterologous interactions usually lead to cross-inhibition, resulting in natural quorum-sensing interference. To gain insight into the mechanisms responsible for these phenomena at the level of the receptor, we used random mutagenesis to isolate variants of Staphylococcus aureus AgrC-I with constitutive activity. Constitutive mutations in the sensor domain of the receptor were localized to the last transmembrane helix, whereas those in the histidine kinase domain were mostly clustered to a region near the phosphorylation site histidine. Analysis of these mutants with a range of noncognate AIPs revealed that inhibition is manifested by inverse agonism in certain heterologous pairings and by neutral antagonism in others. In addition, we isolated and characterized an AgrC sensor domain mutant with dramatically broadened activation specificity and reduced sensitivity to inhibition, identifying a single amino acid as a critical determinant of ligand-mediated inhibition. These results suggest that certain noncognate AIPs stabilize an inhibitory receptor conformation that may be a critical feature of the ligand-receptor interaction not initially appreciated in previous analyses of agr inhibition
PMCID:2633565
PMID: 19147840
ISSN: 1091-6490
CID: 92775
Phage-mediated intergeneric transfer of toxin genes
Because bacteriophages generally parasitize only closely related bacteria, it is assumed that phage-mediated genetic exchange occurs primarily within species. Here we report that staphylococcal pathogenenicity islands, containing superantigen genes, and other mobile elements transferred to Listeria monocytogenes at the same high frequencies as they transfer within Staphylococcus aureus. Several staphylococcal phages transduced L. monocytogenes but could not form plaques. In an experiment modeling phage therapy for bovine mastitis, we observed pathogenicity island transfer between S. aureus and L. monocytogenes in raw milk. Thus, phages may participate in a far more expansive network of genetic information exchange among bacteria of different species than originally thought, with important implications for the evolution of human pathogens
PMID: 19119236
ISSN: 1095-9203
CID: 93388
Intravital two-photon microscopy as a method to analyze the cellular immune response during staphylococcus aureus induced abscess development [Meeting Abstract]
Staphylococcus (S.) aureus is a gram-positive bacterium, which causes local and systemic infections in humans and animals. The immune response to this pathogen typically leads to the development of abscesses in the skin and in inner organs. The cavity of the abscess is filled with neutrophils and is surrounded by macrophages, T cells and a fibrinous capsule. Little is known about the cell migration that takes place during abscess formation after S. aureus infection. Here, we describe an experimental system of intravital two-photon microscopy that allows for studying host-pathogen interactions during abscess development in the skin and in the kidney. Different fluorescent proteins (FP) were expressed in S. aureus in order to visualize the microorganisms. The corresponding genes were adapted to the codon usage of gram-positive bacteria and expressed from a plasmid under the control of the agr P3 or the sarA P1 promoter. Furthermore, the genes and the promoters could be stably integrated into the bacterial chromosome at the phage-related pathogenicity island SaPI1 site. The reporter constructs could then be transferred successfully via phage transduction into the commonly used virulent S. aureus strains RN6734 and RN9130. In addition, an ovalbumin expressing S. aureus strain was generated, which will be used to visualize and elucidate the role of antigen-specific T cells during abscess development. LysM-eGFP knock-in mice, CD11c-YFP and OTII transgenic mice will be used to visualize the recruitment of neutrophils, dendritic cells, and T cells to the site of abscess formation. Initial studies in C57BL/6 wild type mice revealed the presence of FP+ S. aureus in the subcapsular blood vessels of the kidney immediately after injection, where some bacteria were able to adhere to the endothelium. Interestingly, accumulation of pathogens during abscess development was also visible predominantly in the capsular and subcapsular area. Altogether, the described system of intravital microscopy using fluorescent S. aureus strains and reporter mice is a powerful tool, which allows us to follow the immune response during abscess formation in real-time and will provide new insights into the pathogenesis of this clinically highly relevant infection
EMBASE:70347286
ISSN: 0014-2980
CID: 125474
REAL-TIME IN VIVO IMAGING OF LIVER STAGES OF PLASMODIUM YOEL [Meeting Abstract]
ISI:000261644601293
ISSN: 0002-9637
CID: 91862
Prevalence of agr dysfunction among colonizing Staphylococcus aureus strains
Mutations in the staphylococcal virulence regulator gene agr frequently occur during Staphylococcus aureus infection. Whether agr-defective strains are fit for colonization, an important prerequisite for infection, is unknown. Screening by means of assays to detect delta-hemolysin activity and agr autoinducing peptide production indicated that 15 ( approximately 9%) of 160 healthy human subjects were colonized with an agr-defective strain or a mixture of agr-positive and -defective S. aureus strains. The presence of identical agr-defective strains in family members suggests that these strains are transmissible. Additionally, carriage of an agr-defective strain was associated with hospitalization, raising the possibility that such strains may be selected in a nosocomial setting
PMID: 18752431
ISSN: 0022-1899
CID: 93361
