Project description:Q fever is due to Coxiella burnetii, an obligate intracellular bacterium. We investigated the mechanism of establishment of chronic form of the Q fever that mainlym anifested by endocarditis. We showed that patients with acute Q fever and valvulopathy, who have the higher risk to develop an endocarditis, exhibited high levels of circulating apoptotic cells. We further investigated the effect of the uptake of dead cells on the intracellular fate of the bacterium and the immune response of monocytes and macrophages.
Project description:Staphylococcus aureus is an adaptable human pathogen causing life-threatening endocarditis and bacteraemia. Methicillin-resistant S. aureus (MRSA) is alarmingly common, and treatment is confined to last-line antibiotics. Vancomycin is the treatment of choice for MRSA bacteraemia and vancomycin treatment failure is often associated with vancomycin-intermediate S. aureus strains termed VISA. The regulatory 3’ UTR of vigR mRNA contributes to vancomycin tolerance in the clinical VISA isolate JKD6008 and upregulates the lytic transglycosylase IsaA. Using MS2-affinity purification coupled with RNA sequencing (MAPS), we find that the vigR 3' UTR also interacts with mRNAs involved in carbon metabolism, amino acid biogenesis, cell wall biogenesis, and virulence. The vigR 3' UTR was found to repress dapE, a succinyl-diaminopimelate desuccinylase required for lysine and cell wall peptidoglycan synthesis, suggesting a broader role in controlling cell wall metabolism and vancomycin tolerance. Deletion of the vigR 3' UTR increased VISA virulence in a wax moth larvae model, and we find that an isaA mutant is completely attenuated in the larvae model. Sequence and structural analysis of the vigR 3' UTR indicates that the UTR has expanded through the acquisition of Staphylococcus aureus repeat insertions (STAR repeats) that partly contribute sequence for the isaA interaction seed and may functionalise the 3’ UTR. Our findings reveal an extended regulatory network for vigR, uncovering a novel mechanism of regulation of cell wall metabolism and virulence in a clinical S. aureus isolate.
Project description:The precise mechanism and effects of antibiotics in host gene expression and immunomodulation in MRSA infection is unknown. Using a well characterized Methicillin Resistant Staphylococcus aureus (MRSA) isolate USA300 in a murine model of infection, we determined that linezolid and vancomycin induced differential production of bacterial toxins and host cytokines, differences in host gene expression, and differences in immunomodulators during MRSA bloodstream infection. A total of 35 A/J mice, categorized into seven groups (no infection; no infection with linezolid; no infection with vancomycin; 2 hour post-infection (hpi) S. aureus; 24 hpi S. aureus; 24 hpi S. aureus with linezolid; and 24 hpi S. aureus with vancomycin), were used in this study. Mice were injected with USA300 (6 x 106 CFU/g via i.p. route), then intravenously treated with linezolid (25 mg/kg) or vancomycin (25 mg/kg) at 2 hpi. Control and S. aureus infected mice were euthanized at each time point (2 h or 24h) following injection. Whole blood RNA was used for microarray; three cytokines and two S. aureus toxins [PantonValentine Leukocidin (PVL) and alpha hemolysin] were quantified in mouse serum by ELISA. S. aureus CFUs were significantly reduced in blood and kidney after linezolid or vancomycin treatment in S. aureus-infected mice. In vivo IL-1M-NM-2 in mouse serum was significantly reduced in both linezolid (p=0.001) and vancomycin (p=0.006) treated mice compared to untreated ones. IL-6 was significantly reduced only in linezolid treated (p<0.001) but not in vancomycin treated mice. However, another proinflammatory cytokine, TNF-M-NM-1, did not exhibit altered levels in either linezolid or vancomycin treated mice (p=0.3 and p=0.51 respectively). In vivo level of bacterial toxin, Panton-Valentine leukocidin, in mouse serum was significantly reduced only in linezolid treated mice (p=0.02) but not in vancomycin treated mice. There was no significant effect of either treatment in in vivo level of alpha hemolysin production. Unsupervised hierarchical clustering using the gene expression data from 35 microarrays revealed distinct clustering based on infection status and treatment group. Study of the antibiotic-specific difference in gene expression identified the number of genes uniquely expressed in response to S. aureus infection, infection with linezolid treatment, and infection with vancomycin treatment. Pathway associations study for the differentially expressed genes in each comparison group (Control vs. 24 h S. aureus infection, 24 h S. aureus infection vs. 24 h S. aureus linezolid, and 24 h S. aureus infection vs. 24 h S. aureus vancomycin) in mice using Kyoto Encyclopedia of Genes and Genomes (KEGG) identified toll-like receptor signaling pathway to be common to every comparison groups studied. Glycerolipid metabolism pathway was uniquely associated only with linezolid treatment comparison group. The findings of this study provide the evidence that protein synthesis inhibitor like linezolid does a better job in treating MRSA sepsis compared to cell wall acting antibiotics like vancomycin. To identify differences in host gene expression in a murine sepsis model treated with a) linezolid and b) vancomycin, we used whole blood gene expression (RNA) signatures from A/J inbred mice infected with USA 300 MRSA to evaluate differences in host gene expression among mice treated with linezolid and vancomycin. We used 5 RNA samples from MRSA-infected, linezolid- or vancomycin-treated mice. A total of 7 experimental groups have been employed: 1) Uninfected control group: (negative controls). 2) Uninfected, linezolid-treated group: Uninfected, linezolid-treated mice. 3) Uninfected vancomycin-treated group: Uninfected, vancomycin-treated mice. 4) Infected control group (positive control 2 h) MRSA-infected, untreated mice. 5) Infected control group (positive control 24 h): MRSA-infected, untreated mice. 6) Infected linezolid group: MRSA-infected, linezolid-treated mice. 7) Infected vancomycin group: MRSA-infected, vancomycin-treated mice.
Project description:Background: Telavancin is a novel semi-synthetic lipoglycopeptide derivative of vancomycin with a decylaminoethyl side chain that is active against Gram-positive bacteria including Staphylococcus aureus strains resistant to methicillin or vancomycin. This study describes transcriptome alterations in S. aureus strain ATCC29213 treated with telavancin for 15 min and 60 min in comparing with other agents treatment, including vancomycin, enduracidin, m-chlorophenylhydrazone.