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1.  Dusp3 and Psme3 Are Associated with Murine Susceptibility to Staphylococcus aureus Infection and Human Sepsis 
PLoS Pathogens  2014;10(6):e1004149.
Using A/J mice, which are susceptible to Staphylococcus aureus, we sought to identify genetic determinants of susceptibility to S. aureus, and evaluate their function with regard to S. aureus infection. One QTL region on chromosome 11 containing 422 genes was found to be significantly associated with susceptibility to S. aureus infection. Of these 422 genes, whole genome transcription profiling identified five genes (Dcaf7, Dusp3, Fam134c, Psme3, and Slc4a1) that were significantly differentially expressed in a) S. aureus –infected susceptible (A/J) vs. resistant (C57BL/6J) mice and b) humans with S. aureus blood stream infection vs. healthy subjects. Three of these genes (Dcaf7, Dusp3, and Psme3) were down-regulated in susceptible vs. resistant mice at both pre- and post-infection time points by qPCR. siRNA-mediated knockdown of Dusp3 and Psme3 induced significant increases of cytokine production in S. aureus-challenged RAW264.7 macrophages and bone marrow derived macrophages (BMDMs) through enhancing NF-κB signaling activity. Similar increases in cytokine production and NF-κB activity were also seen in BMDMs from CSS11 (C57BL/6J background with chromosome 11 from A/J), but not C57BL/6J. These findings suggest that Dusp3 and Psme3 contribute to S. aureus infection susceptibility in A/J mice and play a role in human S. aureus infection.
Author Summary
Staphylococcus aureus causes life-threatening infections in humans. Host genetic determinants influence the outcome of S. aureus infection, yet are poorly understood. Susceptible A/J and resistant C57BL/6J mice provide a unique platform to study the genetic difference responsible for variable host response to S. aureus infection. We showed that chromosome 11 in A/J was responsible for susceptibility to S. aureus. We further identified a QTL locus on Chromosome 11 significantly associated with S. aureus susceptibility. Five genes in the QTL (Dcaf7, Dusp3, Fam134c, Psme3, and Slc4a1) were significantly differently expressed in a) susceptible vs. resistant mice, and b) humans with S. aureus blood stream infection vs. healthy human subjects. Three genes (Dusp3, Psme3, and Dcaf7) were down-regulated in susceptible A/J mice. siRNA-mediated knockdown of Dusp3 and Psme3 in bone marrow derived macrophage (BMDMs) significantly enhanced cytokine responses through NF-κB activity upon S. aureus challenge in a pattern that was also present in S. aureus-challenged BMDMs from susceptible CSS11 (chr. 11 from A/J but otherwise C57BL/6J) mice, but not resistant C57BL/6J mice. These findings suggest that Dusp3 and Psme3 contribute to S. aureus infection susceptibility in A/J mice and play a role in human S. aureus infection.
doi:10.1371/journal.ppat.1004149
PMCID: PMC4047107  PMID: 24901344
2.  A genome-wide association study of variants associated with acquisition of Staphylococcus aureus bacteremia in a healthcare setting 
Background
Humans vary in their susceptibility to acquiring Staphylococcus aureus infection, and research suggests that there is a genetic basis for this variability. Several recent genome-wide association studies (GWAS) have identified variants that may affect susceptibility to infectious diseases, demonstrating the potential value of GWAS in this arena.
Methods
We conducted a GWAS to identify common variants associated with acquisition of S. aureus bacteremia (SAB) resulting from healthcare contact. We performed a logistic regression analysis to compare patients with healthcare contact who developed SAB (361 cases) to patients with healthcare contact in the same hospital who did not develop SAB (699 controls), testing 542,410 SNPs and adjusting for age (by decade), sex, and 6 significant principal components from our EIGENSTRAT analysis. Additionally, we evaluated the joint effect of the host and pathogen genomes in association with severity of SAB infection via logistic regression, including an interaction of host SNP with bacterial genotype, and adjusting for age (by decade), sex, the 6 significant principal components, and dialysis status. Bonferroni corrections were applied in both analyses to control for multiple comparisons.
Results
Ours is the first study that has attempted to evaluate the entire human genome for variants potentially involved in the acquisition or severity of SAB. Although this study identified no common variant of large effect size to have genome-wide significance for association with either the risk of acquiring SAB or severity of SAB, the variant (rs2043436) most significantly associated with severity of infection is located in a biologically plausible candidate gene (CDON, a member of the immunoglobulin family) and may warrant further study.
Conclusions
The genetic architecture underlying SAB is likely to be complex. Future investigations using larger samples, narrowed phenotypes, and advances in both genotyping and analytical methodologies will be important tools for identifying causative variants for this common and serious cause of healthcare-associated infection.
doi:10.1186/1471-2334-14-83
PMCID: PMC3928605  PMID: 24524581
Genomics; Genome-wide association study; Case–control study; Staphylococcus aureus; Bacteremia; Gram-positive bacterial infections; Polymorphism, single-nucleotide; Infections; Nosocomial; Cross infection
3.  Microparticles from Kidney-Derived Mesenchymal Stem Cells Act as Carriers of Proangiogenic Signals and Contribute to Recovery from Acute Kidney Injury 
PLoS ONE  2014;9(2):e87853.
We recently demonstrated the use of in vitro expanded kidney-derived mesenchymal stem cells (KMSC) protected peritubular capillary endothelial cells in acute renal ischemia-reperfusion injury. Herein, we isolated and characterized microparticles (MPs) from KMSC. We investigated their in vitro biologic effects on human endothelial cells and in vivo renoprotective effects in acute ischemia-reperfusion renal injury. MPs were isolated from the supernatants of KMSC cultured in anoxic conditions in serum-deprived media for 24 hours. KMSC-derived MPs demonstrated the presence of several adhesion molecules normally expressed on KMSC membranes, such as CD29, CD44, CD73, α4, 5, and 6 integrins. Quantitative real time PCR confirmed the presence of 3 splicing variants of VEGF-A (120, 164, 188), bFGF and IGF-1 in isolated MPs. MPs labeled with PKH26 red fluorescence dye were incorporated by cultured human umbilical vein endothelial cells (HUVEC) via surface molecules such as CD44, CD29, and α4, 5, and 6 integrins. MP dose dependently improved in vitro HUVEC proliferation and promoted endothelial tube formation on growth factor reduced Matrigel. Moreover, apoptosis of human microvascular endothelial cell was inhibited by MPs. Administration of KMSC-derived MPs into mice with acute renal ischemia was followed by selective engraftment in ischemic kidneys and significant improvement in renal function. This was achieved by improving proliferation, of peritubular capillary endothelial cell and amelioration of peritubular microvascular rarefaction. Our results support the hypothesis that KMSC-derived MPs may act as a source of proangiogenic signals and confer renoprotective effects in ischemic kidneys.
doi:10.1371/journal.pone.0087853
PMCID: PMC3913695  PMID: 24504266
4.  Host Gene Expression Profiling and In Vivo Cytokine Studies to Characterize the Role of Linezolid and Vancomycin in Methicillin-Resistant Staphylococcus aureus (MRSA) Murine Sepsis Model 
PLoS ONE  2013;8(4):e60463.
Linezolid (L), a potent antibiotic for Methicillin Resistant Staphylococcus aureus (MRSA), inhibits bacterial protein synthesis. By contrast, vancomycin (V) is a cell wall active agent. Here, we used a murine sepsis model to test the hypothesis that L treatment is associated with differences in bacterial and host characteristics as compared to V. Mice were injected with S. aureus USA300, and then intravenously treated with 25 mg/kg of either L or V at 2 hours post infection (hpi). In vivo alpha-hemolysin production was reduced in both L and V-treated mice compared to untreated mice but the reduction did not reach the statistical significance [P = 0.12 for L; P = 0.70 for V). PVL was significantly reduced in L-treated mice compared to untreated mice (P = 0.02). However the reduction of in vivo PVL did not reach the statistical significance in V- treated mice compared to untreated mice (P = 0.27). Both antibiotics significantly reduced IL-1β production [P = 0.001 for L; P = 0.006 for V]. IL-6 was significantly reduced with L but not V antibiotic treatment [P<0.001 for L; P = 0.11 for V]. Neither treatment significantly reduced production of TNF-α. Whole-blood gene expression profiling showed no significant effect of L and V on uninfected mice. In S. aureus-infected mice, L altered the expression of a greater number of genes than V (95 vs. 42; P = 0.001). Pathway analysis for the differentially expressed genes identified toll-like receptor signaling pathway to be common to each S. aureus-infected comparison. Expression of immunomodulatory genes like Cxcl9, Cxcl10, Il1r2, Cd14 and Nfkbia was different among the treatment groups. Glycerolipid metabolism pathway was uniquely associated with L treatment in S. aureus infection. This study demonstrates that, as compared to V, treatment with L is associated with reduced levels of toxin production, differences in host inflammatory response, and distinct host gene expression characteristics in MRSA sepsis.
doi:10.1371/journal.pone.0060463
PMCID: PMC3614971  PMID: 23565251
5.  Gene Expression-Based Classifiers Identify Staphylococcus aureus Infection in Mice and Humans 
PLoS ONE  2013;8(1):e48979.
Staphylococcus aureus causes a spectrum of human infection. Diagnostic delays and uncertainty lead to treatment delays and inappropriate antibiotic use. A growing literature suggests the host’s inflammatory response to the pathogen represents a potential tool to improve upon current diagnostics. The hypothesis of this study is that the host responds differently to S. aureus than to E. coli infection in a quantifiable way, providing a new diagnostic avenue. This study uses Bayesian sparse factor modeling and penalized binary regression to define peripheral blood gene-expression classifiers of murine and human S. aureus infection. The murine-derived classifier distinguished S. aureus infection from healthy controls and Escherichia coli-infected mice across a range of conditions (mouse and bacterial strain, time post infection) and was validated in outbred mice (AUC>0.97). A S. aureus classifier derived from a cohort of 94 human subjects distinguished S. aureus blood stream infection (BSI) from healthy subjects (AUC 0.99) and E. coli BSI (AUC 0.84). Murine and human responses to S. aureus infection share common biological pathways, allowing the murine model to classify S. aureus BSI in humans (AUC 0.84). Both murine and human S. aureus classifiers were validated in an independent human cohort (AUC 0.95 and 0.92, respectively). The approach described here lends insight into the conserved and disparate pathways utilized by mice and humans in response to these infections. Furthermore, this study advances our understanding of S. aureus infection; the host response to it; and identifies new diagnostic and therapeutic avenues.
doi:10.1371/journal.pone.0048979
PMCID: PMC3541361  PMID: 23326304
6.  Haplotype Association Mapping Identifies a Candidate Gene Region in Mice Infected With Staphylococcus aureus 
G3: Genes|Genomes|Genetics  2012;2(6):693-700.
Exposure to Staphylococcus aureus has a variety of outcomes, from asymptomatic colonization to fatal infection. Strong evidence suggests that host genetics play an important role in susceptibility, but the specific host genetic factors involved are not known. The availability of genome-wide single nucleotide polymorphism (SNP) data for inbred Mus musculus strains means that haplotype association mapping can be used to identify candidate susceptibility genes. We applied haplotype association mapping to Perlegen SNP data and kidney bacterial counts from Staphylococcus aureus-infected mice from 13 inbred strains and detected an associated block on chromosome 7. Strong experimental evidence supports the result: a separate study demonstrated the presence of a susceptibility locus on chromosome 7 using consomic mice. The associated block contains no genes, but lies within the gene cluster of the 26-member extended kallikrein gene family, whose members have well-recognized roles in the generation of antimicrobial peptides and the regulation of inflammation. Efficient mixed-model association (EMMA) testing of all SNPs with two alleles and located within the gene cluster boundaries finds two significant associations: one of the three polymorphisms defining the associated block and one in the gene closest to the block, Klk1b11. In addition, we find that 7 of the 26 kallikrein genes are differentially expressed between susceptible and resistant mice, including the Klk1b11 gene. These genes represent a promising set of candidate genes influencing susceptibility to Staphylococcus aureus.
doi:10.1534/g3.112.002501
PMCID: PMC3362298  PMID: 22690378
host genetic susceptibility; infectious disease; kallikrein gene family
7.  Panton-Valentine Leukocidin Is Not the Primary Determinant of Outcome for Staphylococcus aureus Skin Infections: Evaluation from the CANVAS Studies 
PLoS ONE  2012;7(5):e37212.
The impact of Panton-Valentine leukocidin (PVL) on the severity of complicated skin and skin structure infections (cSSSI) caused by Staphylococcus aureus is controversial. We evaluated potential associations between clinical outcome and PVL presence in both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolates from patients enrolled in two large, multinational phase three clinical trials assessing ceftaroline fosamil for the treatment of cSSSI (the CANVAS 1 and 2 programs). Isolates from all microbiologically evaluable patients with monomicrobial MRSA or MSSA infections (n = 473) were genotyped by PCR for pvl and underwent pulsed-field gel electrophoresis (PFGE). Genes encoding pvl were present in 266/473 (56.2%) isolates. Infections caused by pvl-positive S. aureus were associated with younger patient age, North American acquisition, and presence of major abscesses (P<0.001 for each). Cure rates of patients infected with pvl-positive and pvl-negative S. aureus were similar overall (93.6% versus 92.8%; P = 0.72), and within MRSA-infected (94.5% vs. 93.1%; P = 0.67) and MSSA-infected patients (92.2% vs. 92.7%; P = 1.00). This finding persisted after adjustment for multiple patient characteristics. Outcomes were also similar when USA300 PVL+ and non-USA300 PVL+ infections were compared. The results of this contemporary, international study suggest that pvl presence was not the primary determinant of outcome in patients with cSSSI due to either MRSA or MSSA.
doi:10.1371/journal.pone.0037212
PMCID: PMC3356380  PMID: 22623995
8.  Effect on Glycemic, Blood Pressure, and Lipid Control according to Education Types 
Diabetes & Metabolism Journal  2011;35(6):580-586.
Background
Diabetes self-management education and reinforcement are important for effective management of the disease. We investigated the effectiveness of interactive small-group education on glycemic, blood pressure, and lipid levels.
Methods
For this study, 207 type 2 diabetes patients with suboptimal glycemic control (HbA1c levels >6.5%) were enrolled. The conventional education group received an existing education program from April to November in 2006, and the interactive education group received a new small-group education program from December 2006 to July 2007. The two groups were comparatively analyzed for changes in blood sugar, glycated hemoglobin, lipid, and blood pressure at baseline, 3, 6, and 12 months and the proportion of patients achieving target goals at 12 months.
Results
After 12 months of follow-up, HbA1c levels in the interactive education group were significantly lower than in the conventional education group (6.7% vs. 6.4%, P<0.001). Fasting and 2 hour postprandial glucose concentrations, total cholesterol, and low density lipoprotein cholesterol were significantly lower in the interactive education group than in the conventional education group. The proportion of patients that achieved target goals was significantly higher in the interactive education group.
Conclusion
The small-group educational method improved and re-established the existing group educational method. This finding suggests that the importance of education appears to be related to the method by which it is received rather than the education itself. Thus, the use of small-group educational methods to supplement existing educational methods established for diverse age levels should be considered in the future.
doi:10.4093/dmj.2011.35.6.580
PMCID: PMC3253968  PMID: 22247900
Achievement; Diabetes mellitus; Education
9.  Two Genes on A/J Chromosome 18 Are Associated with Susceptibility to Staphylococcus aureus Infection by Combined Microarray and QTL Analyses 
PLoS Pathogens  2010;6(9):e1001088.
Although it has recently been shown that A/J mice are highly susceptible to Staphylococcus aureus sepsis as compared to C57BL/6J, the specific genes responsible for this differential phenotype are unknown. Using chromosome substitution strains (CSS), we found that loci on chromosomes 8, 11, and 18 influence susceptibility to S. aureus sepsis in A/J mice. We then used two candidate gene selection strategies to identify genes on these three chromosomes associated with S. aureus susceptibility, and targeted genes identified by both gene selection strategies. First, we used whole genome transcription profiling to identify 191 (56 on chr. 8, 100 on chr. 11, and 35 on chr. 18) genes on our three chromosomes of interest that are differentially expressed between S. aureus-infected A/J and C57BL/6J. Second, we identified two significant quantitative trait loci (QTL) for survival post-infection on chr. 18 using N2 backcross mice (F1 [C18A]×C57BL/6J). Ten genes on chr. 18 (March3, Cep120, Chmp1b, Dcp2, Dtwd2, Isoc1, Lman1, Spire1, Tnfaip8, and Seh1l) mapped to the two significant QTL regions and were also identified by the expression array selection strategy. Using real-time PCR, 6 of these 10 genes (Chmp1b, Dtwd2, Isoc1, Lman1, Tnfaip8, and Seh1l) showed significantly different expression levels between S. aureus-infected A/J and C57BL/6J. For two (Tnfaip8 and Seh1l) of these 6 genes, siRNA-mediated knockdown of gene expression in S. aureus–challenged RAW264.7 macrophages induced significant changes in the cytokine response (IL-1 β and GM-CSF) compared to negative controls. These cytokine response changes were consistent with those seen in S. aureus-challenged peritoneal macrophages from CSS 18 mice (which contain A/J chromosome 18 but are otherwise C57BL/6J), but not C57BL/6J mice. These findings suggest that two genes, Tnfaip8 and Seh1l, may contribute to susceptibility to S. aureus in A/J mice, and represent promising candidates for human genetic susceptibility studies.
Author Summary
Staphylococcus aureus has a wide spectrum of human infection, ranging from asymptomatic nasal carriage to overwhelming sepsis and death. Mouse models offer an attractive strategy for investigating complex diseases such as S. aureus infections. A/J mice are highly susceptible to S. aureus infection compared with C57BL/6J mice. We showed that genes on chromosomes 8, 11, and 18 in A/J are responsible for susceptibility to S. aureus by using chromosome substitution strains (CSS). From the ∼4200 genes on these three chromosomes, we identified 191 which were differentially expressed between A/J and C57BL/6J when challenged with S. aureus. Next, we identified two significant QTLs on chromosome 18 that are associated with susceptibility to S. aureus infection in N2 backcross mice. Ten genes (March3, Cep120, Chmp1b, Dcp2, Dtwd2, Isoc1, Lman1, Spire1, Tnfaip8, and Seh1l) mapped to the two significant QTLs and were differentially expressed between A/J and C57BL/6J. One gene on each QTL, Tnfaip8 and Seh1l, affected expression of cytokines in mouse macrophages exposed to S. aureus. These cytokine response patterns were consistent with those seen in S. aureus-challenged peritoneal macrophages from CSS 18, but not C57BL/6J. Tnfaip8 and Seh1l are strong candidates for genes influencing susceptibility to S. aureus of A/J mice.
doi:10.1371/journal.ppat.1001088
PMCID: PMC2932726  PMID: 20824097
10.  Critical Role of NOD2 in Regulating the Immune Response to Staphylococcus aureus▿  
Infection and Immunity  2009;77(4):1376-1382.
NOD2 (the nucleotide-binding oligomerization domain containing protein 2) is known to be involved in host recognition of bacteria, although its role in the host response to Staphylococcus aureus infection is unknown. NOD2-deficient (Nod2−/−) mice and wild-type (WT) littermate controls were injected intraperitoneally with S. aureus suspension (107 bacteria/g of body weight), and their survival was monitored. Cultured bone marrow-derived neutrophils were harvested from Nod2−/− and WT mice and tested for cytokine production and phagocytosis. Compared to WT mice, Nod2−/− mice were significantly more susceptible to S. aureus infection (median survival of 1.5 days versus >5 days; P = 0.003) and had a significantly higher bacterial tissue burden. Cultured bone marrow-derived neutrophils from Nod2−/− and WT mice had similar levels of peritoneal neutrophil recruitment and intracellular killing, but bone marrow-derived neutrophils from Nod2−/− mice had significantly reduced ability to internalize fluorescein-labeled S. aureus. Nod2−/− mice had significantly higher levels of Th1-derived cytokines in serum (tumor necrosis factor alpha, gamma interferon, and interleukin-2 [IL-2]) compared to WT mice, whereas the levels of Th2-derived cytokines (IL-1β, IL-4, IL-6, and IL-10) were similar in Nod2−/− and WT mice. Thus, mice deficient in NOD2 are more susceptible to S. aureus. Increased susceptibility is due in part to defective neutrophil phagocytosis, elevated serum levels of Th1 cytokines, and a higher bacterial tissue burden.
doi:10.1128/IAI.00940-08
PMCID: PMC2663139  PMID: 19139201
11.  Identification of an Iron-Regulated Hemin-Binding Outer Membrane Protein, HupO, in Vibrio fluvialis: Effects on Hemolytic Activity and the Oxidative Stress Response  
Infection and Immunity  2005;73(2):722-729.
In pathogenic bacteria, iron acquisition is important for colonization and proliferation in the host under iron-limited conditions. The ability of Vibrio spp. to acquire iron is often critical to their virulence, causing gastroenteritis or excessive watery diarrhea in humans. In the study described here, we cloned the 2,100-bp heme utilization protein gene hupO in Vibrio fluvialis. HupO had high homology to iron-regulated outer membrane receptor proteins in Vibrio sp. and contained motifs that are common to bacterial heme receptors, including a consensus TonB box, a FRAP domain, and an NPNL domain. To characterize the hemin-binding activity of HupO, we purified the recombinant HupO protein (rHupO) from Escherichia coli by using an overexpression system. HupO was found to bind to hemin but not to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting demonstrated that the 77-kDa outer membrane protein HupO of V. fluvialis was induced under iron-restricted conditions. We constructed a hupO mutant, HP1, to investigate the biochemical function of HupO in V. fluvialis. The hemolytic activity of HP1 was reduced compared to that of wild-type cells and, when exposed to hydrogen peroxide, significantly lower numbers of HP1 survived than was the case in the wild type. These results suggest that HupO is associated with virulence expression in V. fluvialis through stimulation of hemolysin production and resistance to oxidative stress. In experimentally infected mice, the 50% lethal dose value of the wild-type was lower than that of the mutant, HP1.
doi:10.1128/IAI.73.2.722-729.2005
PMCID: PMC546946  PMID: 15664910
12.  Frequency of Mutations Conferring Resistance to Nucleoside Reverse Transcriptase Inhibitors in Human Immunodeficiency Virus Type 1-Infected Patients in Korea 
Journal of Clinical Microbiology  2002;40(4):1319-1325.
A nested PCR and direct sequencing methods were used to define human immunodeficiency virus type 1(HIV-1) reverse transcriptase codons 41 to 219 in DNA from 127 peripheral blood mononuclear cell samples obtained from 35 patients treated with nucleoside reverse transcriptase inhibitors (NRTI). The follow-up period after the initiation of NRTI therapy was 61.8 ± 31 months (mean and standard deviation). In addition to NRTI therapy, 32 of 35 patients were simultaneously treated with Korean red ginseng. The annual decrease in the CD4+ T-cell count over 5 years was 13.2/μl. Twenty-eight (80%) of the 35 patients had mutations conferring resistance to NRTI. The frequencies of K70R, T215S/Y/F (i.e., mutation of T at codon 215 to S, Y, or F), D67N/E, K219Q, T69N/S/A, M41L, and L210W mutations conferring resistance to zidovudine were 57.6, 36.4, 36.4, 27.2, 24.2, 21.2, and 12.1%, respectively. Mutations conferring resistance to didanosine and lamivudine were detected in 2 (L74V and M184I; 14.2%) of 11 patients tested and in 4 (M184V; 57%) of 7 patients tested, respectively. In particular, the frequency of T69N/S/A increased sharply after more than 48 months of zidovudine monotherapy. However, Q151M was not detected. As the first report on the frequency of NRTI resistance mutations in Korea, our data suggest that genotypic antiretroviral drug testing should be considered for the design of better drug regimens to improve the management of HIV-1-infected patients.
doi:10.1128/JCM.40.4.1319-1325.2002
PMCID: PMC140365  PMID: 11923351

Results 1-12 (12)