Biomedical subjects

Steven J Projan

Publications and source records attributed to Steven J Projan.

At least 19 recordsLinked to original sources

PubMed2006

Staphylococcal vaccines and immunotherapy: to dream the impossible dream?

Staphylococcal infections not only remain an important cause of morbidity and mortality in both the community and the clinic, but the emergence of a global pandemic of community-associated methicillin-resistant Staphylococcus aureus, involving what purports to be a more virulent strain of this organism, has also led several in the infectious disease community to call for improved disease prevention strategies (in addition to novel therapeutics) in what could be thought of as the microbiological version of pre-emption. In this case, Staphylococcus aureus possesses "weapons of mass destruction" and appears to be using them effectively as part of anti-immunization insurgency.

Adhesins, Bacterial↗

PubMed2006

Transcriptional profiling of a Staphylococcus aureus clinical isolate and its isogenic agr and sarA mutants reveals global differences in comparison to the laboratory strain RN6390.

The production of Staphylococcus aureus virulence factors is under the control of complex regulatory circuits. Most studies aimed at defining these regulatory networks have focused on derivatives of the strain NCTC 8325, most notably RN6390. However, all NCTC 8325 derivatives, including RN6390, possess an 11 bp deletion in rsbU. This deletion renders NCTC 8325 derivatives naturally sigma-factor-B deficient. Recent studies have shown that RN6390 is also deficient, in comparison to clinical isolates, with respect to biofilm formation, a process which is important for both pathogenesis and antimicrobial resistance. Based on these considerations, the authors carried out genome-scale transcriptional profiling, comparing RN6390 with the virulent rsbU-positive clinical isolate UAMS-1. The results revealed significant genome-wide differences in expression patterns between RN6390 and UAMS-1, and suggested that the overall transcriptional profile of UAMS-1 is geared toward expression of factors that promote colonization and biofilm formation. In contrast, the transcriptional profile of RN6390 was heavily influenced by RNAIII expression, resulting in a phenotype characterized by increased production of exoproteins, and decreased capacity to form a biofilm. The greater influence of agr in RN6390 relative to UAMS-1 was also evident when the transcriptional profile of UAMS-1 was compared with that of its isogenic sarA and agr mutants. Specifically, the results indicate that, in contrast to NCTC 8325 derivatives, agr plays a limited role in overall regulation of gene expression in UAMS-1, when compared with sarA. Furthermore, by defining the sarA regulon in a biofilm-positive clinical isolate, and comparing the results with transcriptional profiling experiments defining biofilm-associated gene expression patterns in the same strain, the authors identified a sarA-regulated operon (alsSD) that is also induced in biofilms, and demonstrated that mutation of alsSD results in reduced capacity to form a biofilm.

Adaptation, Physiological↗

PubMed2006

Characterization of the Staphylococcus aureus CidR regulon: elucidation of a novel role for acetoin metabolism in cell death and lysis.

The Staphylococcus aureus cid and lrg operons encode a novel regulatory system that affects murein hydrolase activity, stationary-phase survival and antibiotic tolerance. Expression of the lrgAB operon is positively regulated by a two-component regulatory system encoded by the lytSR operon located immediately upstream to lrgAB. By comparison, the cidABC operon lies downstream from the cidR gene, encoding a protein homologous to the LysR-type family of transcriptional regulators. Transcription analysis of a cidR mutant revealed that CidR enhances cidABC expression in the presence of acetic acid generated by the metabolism of excess glucose. Microarray studies identified additional CidR-regulated operons including the IrgAB and alsSD encoding proteins involved in acetoin production. Surprisingly, Northern blot analyses revealed that cidABC and lrgAB transcription was uninducible in an alsSD mutant grown in the presence of excess glucose, suggesting that the CidR-mediated upregulation of cidABC and lrgAB transcription is dependent on the presence of intact alsSD genes. Zymographic and quantitative analyses of murein hydrolase activity also revealed that disruption of the alsSD genes results in significantly decreased extracellular murein hydrolase activity compared with that of the parental strain, UAMS-1. Furthermore, the alsSD mutant displayed decreased stationary-phase survival relative to UAMS-1, both in the presence and absence of glucose. The results of this study define the CidR regulon and demonstrate that the generation of acetoin is linked to the control of cell death and lysis in S. aureus.

Acetoin↗

PubMed2006

Characterization of the Staphylococcus aureus heat shock, cold shock, stringent, and SOS responses and their effects on log-phase mRNA turnover.

Despite its being a leading cause of nosocomal and community-acquired infections, surprisingly little is known about Staphylococcus aureus stress responses. In the current study, Affymetrix S. aureus GeneChips were used to define transcriptome changes in response to cold shock, heat shock, stringent, and SOS response-inducing conditions. Additionally, the RNA turnover properties of each response were measured. Each stress response induced distinct biological processes, subsets of virulence factors, and antibiotic determinants. The results were validated by real-time PCR and stress-mediated changes in antimicrobial agent susceptibility. Collectively, many S. aureus stress-responsive functions are conserved across bacteria, whereas others are unique to the organism. Sets of small stable RNA molecules with no open reading frames were also components of each response. Induction of the stringent, cold shock, and heat shock responses dramatically stabilized most mRNA species. Correlations between mRNA turnover properties and transcript titers suggest that S. aureus stress response-dependent alterations in transcript abundances can, in part, be attributed to alterations in RNA stability. This phenomenon was not observed within SOS-responsive cells.

Adaptation, Physiological↗

PubMed2006

Transcription Profiling of the mgrA Regulon in Staphylococcus aureus.

MgrA has been shown to affect multiple Staphylococcus aureus genes involved in virulence and antibiotic resistance. To comprehensively identify the target genes regulated by mgrA, we employed a microarray method to analyze the transcription profiles of S. aureus Newman, its isogeneic mgrA mutant, and an MgrA-overproducing derivative. We compared genes that were differentially expressed at exponential or early stationary growth phases. Our results showed that MgrA affected an impressive number of genes, 175 of which were positively regulated and 180 of which were negatively regulated in an mgrA-specific manner. The target genes included all functional categories. The microarray results were validated by real-time reverse transcription-PCR quantitation of a set of selected genes from different functional categories. Our data also indicate that mgrA regulates virulence factors in a fashion analogous to that of the accessory gene regulatory locus (agr). Accordingly, exoproteins are upregulated and surface proteins are downregulated by the regulator, suggesting that mgrA may function in concert with agr. The fact that a large number of genes are regulated by mgrA implies that MgrA is a major global regulator in S. aureus.

Bacterial Proteins↗

PubMed2006

Characterizing the effect of the Staphylococcus aureus virulence factor regulator, SarA, on log-phase mRNA half-lives.

Bacterial pathogens regulate virulence factor expression at both the level of transcription initiation and mRNA processing/turnover. Within Staphylococcus aureus, virulence factor transcript synthesis is regulated by a number of two-component regulatory systems, the DNA binding protein SarA, and the SarA family of homologues. However, little is known about the factors that modulate mRNA stability or influence transcript degradation within the organism. As our entree to characterizing these processes, S. aureus GeneChips were used to simultaneously determine the mRNA half-lives of all transcripts produced during log-phase growth. It was found that the majority of log-phase transcripts (90%) have a short half-life (<5 min), whereas others are more stable, suggesting that cis- and/or trans-acting factors influence S. aureus mRNA stability. In support of this, it was found that two virulence factor transcripts, cna and spa, were stabilized in a sarA-dependent manner. These results were validated by complementation and real-time PCR and suggest that SarA may regulate target gene expression in a previously unrecognized manner by posttranscriptionally modulating mRNA turnover. Additionally, it was found that S. aureus produces a set of stable RNA molecules with no predicted open reading frame. Based on the importance of the S. aureus agr RNA molecule, RNAIII, and small stable RNA molecules within other pathogens, it is possible that these RNA molecules influence biological processes within the organism.

Bacterial Proteins↗

PubMed2006

Characterization of a strain of community-associated methicillin-resistant Staphylococcus aureus widely disseminated in the United States.

A highly stable strain of Staphylococcus aureus with a pulsed-field gel electrophoresis type of USA300 and multilocus sequence type 8 has been isolated from patients residing in diverse geographic regions of the United States. This strain, designated USA300-0114, is a major cause of skin and soft tissue infections among persons in community settings, including day care centers and correctional facilities, and among sports teams, Native Americans, men who have sex with men, and military recruits. The organism is typically resistant to penicillin, oxacillin, and erythromycin (the latter mediated by msrA) and carries SCCmec type IVa. This strain is variably resistant to tetracycline [mediated by tet(K)]; several recent isolates have decreased susceptibility to fluoroquinolones. S. aureus USA300-0114 harbors the genes encoding the Panton-Valentine leucocidin toxin. DNA sequence analysis of the direct repeat units within the mec determinant of 30 USA300-0114 isolates revealed differences in only a single isolate. Plasmid analysis identified a common 30-kb plasmid that hybridized with blaZ and msrA probes and a 3.1-kb cryptic plasmid. A 4.3-kb plasmid encoding tet(K) and a 2.6-kb plasmid encoding ermC were observed in a few isolates. DNA microarray analysis was used to determine the genetic loci for a series of virulence factors and genes associated with antimicrobial resistance. Comparative genomics between USA300-0114 and three other S. aureus lineages (USA100, USA400, and USA500) defined a set of USA300-0114-specific genes, which may facilitate the strain's pathogenesis within diverse environments.

Community-Acquired Infections↗

PubMed2005

Differential proteomic analysis of bronchoalveolar lavage fluid in asthmatics following segmental antigen challenge.

Allergic asthma is characterized by persistent airway inflammation and remodeling. Bronchoalveolar lavage conducted with fiberoptic bronchoscopy has been widely used for investigating the pathogenesis of asthma and other lung disorders. Identification of proteins in the bronchoalveolar lavage fluid (BALF) and their expression changes at different stages of asthma could provide further insights into the complex molecular mechanisms involved in this disease. In this report, we describe the first comprehensive differential proteomic analysis of BALF from both asthmatic patients and healthy subjects before and 24 h after segmental allergen challenge. Our proteomic analysis involves affinity depletion of six abundant BALF proteins, SDS-PAGE fractionation, protein in-gel digestion, and subsequent nano-LC-MS/MS analysis in conjunction with database searching for protein identification and semiquantitation. More than 1,500 distinct proteins were identified of which about 10% displayed significant up-regulation specific to the asthmatic patients after segmental allergen challenge. The differentially expressed proteins represent a wide spectrum of functional classes such as chemokines, cytokines, proteases, complement factors, acute phase proteins, monocyte-specific granule proteins, and local matrix proteins, etc. The majority of these protein expression changes are closely associated with many aspects of the pathophysiology of asthma, including inflammation, eosinophilia, airway remodeling, tissue damage and repair, mucus production, and plasma infiltration. Importantly a large portion of these proteins and their expression changes were identified for the first time from BALF, thus providing new insights for finding novel pathological mediators and biomarkers of asthma.

Adult↗

PubMed2005

Use of ribotyping to retrospectively identify methicillin-resistant Staphylococcus aureus isolates from phase 3 clinical trials for tigecycline that are genotypically related to community-associated isolates.

A retrospective study was performed to identify methicillin-resistant Staphylococcus aureus (MRSA) isolates obtained from patients enrolled in phase 3 clinical trials for tigecycline that were genotypically similar to known community-associated MRSA (CA-MRSA) strains. The clinical trials were double-blind comparator studies for complicated skin and skin structure infections or complicated intra-abdominal infections. We obtained 85% of the MRSA isolates from patients with complicated skin and skin structure infections. Using ribotyping, MRSA isolates were compared with well-characterized North American CA-MRSA strains and negative-control hospital-associated (HA) MRSA strains by cluster analysis; 91 of the 173 isolates clustered with two groups of known CA-MRSA strains, 60% of which shared an indistinguishable ribotype. These isolates were subsequently tested for the presence of SCCmec type IV and the Panton-Valentine leukocidin (PVL)-encoding genes as well as susceptibility to clindamycin, characteristics that are typically associated with CA-MRSA; 89 of the 91 isolates carried the type IV SCCmec element and 76 were also positive for the PVL-encoding genes; 73 of these isolates were susceptible to clindamycin. A similar analysis performed on 26 nonclustering isolates identified only four with these characteristics; 89 of the 91 clustering isolates were inhibited by tigecycline at MICs of < or = 0.5 microg/ml. On the basis of clustering information and preliminary genetic characterization, it appears that ribotyping is a useful tool in identifying potential CA-MRSA isolates and 76 MRSA isolates from patients enrolled in the tigecycline phase 3 trials have genetic markers typically associated with CA-MRSA.

Cluster Analysis↗

PubMed2005

A novel MATE family efflux pump contributes to the reduced susceptibility of laboratory-derived Staphylococcus aureus mutants to tigecycline.

Tigecycline, an expanded-broad-spectrum glycylcycline antibiotic is not affected by the classical tetracycline resistance determinants found in Staphylococcus aureus. The in vitro selection of mutants with reduced susceptibility to tigecycline was evaluated for two methicillin-resistant S. aureus strains by serial passage in increasing concentrations of tigecycline. Both strains showed a stepwise elevation in tigecycline MIC over a period of 16 days, resulting in an increase in tigecycline MIC of 16- and 32-fold for N315 and Mu3, respectively. Transcriptional profiling revealed that both mutants exhibited over 100-fold increased expression of a gene cluster, mepRAB (multidrug export protein), encoding a MarR-like transcriptional regulator (mepR), a novel MATE family efflux pump (mepA), and a hypothetical protein of unknown function (mepB). Sequencing of the mepR gene in the mutant strains identified changes that presumably inactivated the MepR protein, which suggested that MepR functions as a repressor of mepA. Overexpression of mepA in a wild-type background caused a decrease in susceptibility to tigecycline and other substrates for MATE-type efflux pumps, although it was not sufficient to confer high-level resistance to tigecycline. Complementation of the mepR defect by overexpressing a wild-type mepR gene reduced mepA transcription and lowered the tigecycline MIC in the mutants. Transcription of tet(M) also increased by over 40-fold in the Mu3 mutant. This was attributed to a deletion in the promoter region of the gene that removed a stem-loop responsible for transcriptional attenuation. However, overexpression of the tet(M) transcript in a tigecycline-susceptible strain was not enough to significantly increase the MIC of tigecycline. These results suggest that the overexpression of mepA but not tet(M) may contribute to decreased susceptibility of tigecycline in S. aureus.

Anti-Bacterial Agents↗

PubMed2005

Comparative genomics of Staphylococcus aureus musculoskeletal isolates.

Much of the research aimed at defining the pathogenesis of Staphylococcus aureus has been done with a limited number of strains, most notably the 8325-4 derivative RN6390. Several lines of evidence indicate that this strain is unique by comparison to clinical isolates of S. aureus. Based on this, we have focused our efforts on two clinical isolates (UAMS-1 and UAMS-601), both of which are hypervirulent in our animal models of musculoskeletal infection. In this study, we used comparative genomic hybridization to assess the genome content of these two isolates relative to RN6390 and each of seven sequenced S. aureus isolates. Our comparisons were done by using an amplicon-based microarray from the Pathogen Functional Genomics Resource Center and an Affymetrix GeneChip that collectively represent the genomes of all seven sequenced strains. Our results confirmed that UAMS-1 and UAMS-601 share specific attributes that distinguish them from RN6390. Potentially important differences included the presence of cna and the absence of isaB, sarT, sarU, and sasG in the UAMS isolates. Among the sequenced strains, the UAMS isolates were most closely related to the dominant European clone EMRSA-16. In contrast, RN6390, NCTC 8325, and COL formed a distinct cluster that, by comparison to the other four sequenced strains (Mu50, N315, MW2, and SANGER-476), was the most distantly related to the UAMS isolates and EMRSA-16.

Adhesins, Bacterial↗

PubMed2004

Hydrophobic acetal and ketal derivatives of mannopeptimycin-alpha and desmethylhexahydromannopeptimycin-alpha: semisynthetic glycopeptides with potent activity against Gram-positive bacteria.

The effect of introducing hydrophobic groups onto the disaccharide portion of the mannopeptimycins has been examined. Under acid-catalyzed conditions dimethyl acetals and ketals react on the terminal mannose of the disaccharide moiety of mannopeptimycin-alpha and the cyclohexylalanyl analogue 2. The preferentially formed monofunctionalized 4,6-acetals and -ketals display potent antibacterial activities against Gram-positive microorganisms, including MRSA, PRSP, and VRE pathogens.

Acetals↗

PubMed2004

Comparison of tetracycline and tigecycline binding to ribosomes mapped by dimethylsulphate and drug-directed Fe2+ cleavage of 16S rRNA.

OBJECTIVES: The new antibiotic tigecycline (9-t-butylglycylamido-minocycline; GAR-936) overcomes most of the known tetracycline resistance mechanisms. Here we analyse its mode of antibiotic action by probing 70S ribosomes of Escherichia coli with dimethylsulphate (DMS) and Fe(2+)-mediated cleavage to identify binding sites of tetracycline and tigecycline. METHODS: Fe(2+)-mediated cleavage makes use of the ability of Fe2+ to replace the Mg2+ ion complexed with tetracyclines. After addition of H2O2, Fe2+ generates short-lived, highly reactive hydroxyl radicals that can cleave RNA close to the tetracycline binding sites. RESULTS: We identified three prominent Fe(2+)-mediated cleavage sites in helices 29 and 34, and in the internal loop of helix 31 of 16S rRNA in the presence of tetracycline or tigecycline. Qualitatively, these sites are modified identically by both antibiotics, but quantitative differences observed in the cleavage intensities indicate that the drugs bind in slightly different orientations. These results are supported by DMS modification, mutational analysis of 16S rRNA and structural modelling of tigecycline at a tetracycline-binding site in the 30S ribosomal subunit. CONCLUSIONS: Both derivatives bind to identical or overlapping sites and probably share the same mode of antibiotic action. The fact that tigecycline overcomes most of the known tetracycline resistance mechanisms is interpreted as a result of steric hindrance due to the large substituent at position 9.

Base Sequence↗

PubMed2004

Effect of srtA and srtB gene expression on the virulence of Staphylococcus aureus in animal models of infection.

OBJECTIVE: The role that the surface proteins anchored by the srtA and srtB gene products play in the ability of Staphylococcus aureus bacteria to establish infection was investigated in several animal models. METHODS: Wild-type and corresponding mutants with deletions of the srtA and/or srtB genes were used in murine acute lethal infection, septic arthritis, kidney infection and rat endocarditis models. RESULTS: The LD(50) of the wild-type and srtB- knockout were comparable and approximately two- to four-fold lower than the required inoculum of the srtA- and srtA-B- strains. This difference was exhibited as a two-fold greater mortality at the highest inoculum. The wild-type strain established arthritic inflammation in over 90% of the animals with a maximum arthritic index of 6.5 by days 17-21. The srtB- knockout was able to cause inflammation in 70-80% of the mice, but with a lower index of 3.0. Both the srtA- and srtA-B- strains appeared to be less virulent in this model with arthritic indices of around 0.5 and only 20% of the animals with inflammation. Strains with the srtA mutation achieved statistically significant lower titres than wild-type in kidneys of mice after intravenous infection. Mean bacterial counts in cardiac vegetations were significantly higher for the wild-type and srtB- strain compared with the srtA- and srtA-B- strains. CONCLUSION: Results from this study substantiate the role of the srtA gene product in the establishment of infections and further studies are warranted to define and exploit this as a target for antimicrobial chemotherapy.

Aminoacyltransferases↗

PubMed2004

Global gene expression in Staphylococcus aureus biofilms.

We previously demonstrated that mutation of the staphylococcal accessory regulator (sarA) in a clinical isolate of Staphylococcus aureus (UAMS-1) results in an impaired capacity to form a biofilm in vitro (K. E. Beenken, J. S. Blevins, and M. S. Smeltzer, Infect. Immun. 71:4206-4211, 2003). In this report, we used a murine model of catheter-based biofilm formation to demonstrate that a UAMS-1 sarA mutant also has a reduced capacity to form a biofilm in vivo. Surprisingly, mutation of the UAMS-1 ica locus had little impact on biofilm formation in vitro or in vivo. In an effort to identify additional loci that might be relevant to biofilm formation and/or the adaptive response required for persistence of S. aureus within a biofilm, we isolated total cellular RNA from UAMS-1 harvested from a biofilm grown in a flow cell and compared the transcriptional profile of this RNA to RNA isolated from both exponential- and stationary-phase planktonic cultures. Comparisons were done using a custom-made Affymetrix GeneChip representing the genomic complement of six strains of S. aureus (COL, N315, Mu50, NCTC 8325, EMRSA-16 [strain 252], and MSSA-476). The results confirm that the sessile lifestyle associated with persistence within a biofilm is distinct by comparison to the lifestyles of both the exponential and postexponential phases of planktonic culture. Indeed, we identified 48 genes in which expression was induced at least twofold in biofilms over expression under both planktonic conditions. Similarly, we identified 84 genes in which expression was repressed by a factor of at least 2 compared to expression under both planktonic conditions. A primary theme that emerged from the analysis of these genes is that persistence within a biofilm requires an adaptive response that limits the deleterious effects of the reduced pH associated with anaerobic growth conditions.

Adaptation, Physiological↗

PubMed2004

Effect of mild acid on gene expression in Staphylococcus aureus.

During staphylococcal growth in glucose-supplemented medium, the pH of a culture starting near neutrality typically decreases by about 2 units due to the fermentation of glucose. Many species can comfortably tolerate the resulting mildly acidic conditions (pH, approximately 5.5) by mounting a cellular response, which serves to defend the intracellular pH and, in principle, to modify gene expression for optimal performance in a mildly acidic infection site. In this report, we show that changes in staphylococcal gene expression formerly thought to represent a glucose effect are largely the result of declining pH. We examine the cellular response to mild acid by microarray analysis and define the affected gene set as the mild acid stimulon. Many of the genes encoding extracellular virulence factors are affected, as are genes involved in regulation of virulence factor gene expression, transport of sugars and peptides, intermediary metabolism, and pH homeostasis. Key results are verified by gene fusion and Northern blot hybridization analyses. The results point to, but do not define, possible regulatory pathways by which the organism senses and responds to a pH stimulus.

Bacterial Proteins↗

PubMed2004

Small molecules for small minds? The case for biologic pharmaceuticals.

Biologic pharmaceuticals are gaining in both market share and clinical utility compared with small molecule therapeutics. This market growth is, in part, reflective of a field of science entering its toddlerhood, where with increased maturity, both development timelines and costs of manufacturing for these complex molecules will decrease, further enhancing the profitability side of the equation. Although a firm understanding of the rules governing toxicity (especially antibody responses to therapeutic proteins) remains to be defined, it is clear that proteins are less prone to much of the idiosyncratic toxicity associated with small molecule drug candidates. Proteins are disadvantaged in that they are unlikely to find much use in targeting intercellular processes; however, they have clear strengths over small molecules in targeting protein-protein interactions and the specific targeting of surface features of particular cells (e.g., in oncology). As each aspect of protein pharmaceutical technology advances, it is clear that this will be the major area for growth in the industry over the next decade.

Antibodies, Monoclonal↗

PubMed2003

Inactivation of mprF affects vancomycin susceptibility in Staphylococcus aureus.

A chemically generated mutant of Staphylococcus aureus RN4220, GC6668, was isolated that had a fourfold increase in resistance to vancomycin. This phenotype reverted back to susceptibility by insertional mutagenesis with Tn917. In a selected set of revertants, Tn917 insertion was mapped to a unique chromosomal region upstream of mprF, a recently described gene that determines staphylococcal resistance to several host defense peptides. The genetic linkage between the vancomycin susceptibility and Tn917 insertion was then confirmed by transduction backcrosses into both GC6668 and GISA isolates, MER-S12 and HT2002 0127. Northern blot analysis, insertional inactivation and complementation experiments showed that mprF mediates vancomycin susceptibility in S. aureus. The inactivation of mprF by Tn917 insertion in HT2002 0127 caused a significant increase in the binding of vancomycin to the cell membranes. This observation serves as a likely mechanism of the increased vancomycin susceptibility associated with mprF inactivation.

Aminoacyltransferases↗