Transcriptomic Analysis of Peritoneal Cells in a Mouse Model of Sepsis: Confirmatory and Novel Results in Early and Late Sepsis.

Background The events leading to sepsis start with an invasive infection of a primary organ of the body followed by an overwhelming systemic response. Intra-abdominal infections are the second most common cause of sepsis. Peritoneal fluid is the primary site of infection in these cases. A microarray-based approach was used to study the temporal changes in cells from the peritoneal cavity of septic mice and to identify potential biomarkers and therapeutic targets for this subset of sepsis patients. Results We conducted microarray analysis of the peritoneal cells of mice infected with a non-pathogenic strain of Escherichia coli. Differentially expressed genes were identified at two early (1 h, 2 h) and one late time point (18 h). A multiplexed bead array analysis was used to confirm protein expression for several cytokines which showed differential expression at different time points based on the microarray data. Gene Ontology based hypothesis testing identified a positive bias of differentially expressed genes associated with cellular development and cell death at 2 h and 18 h respectively. Most differentially expressed genes common to all 3 time points had an immune response related function, consistent with the observation that a few bacteria are still present at 18 h. Conclusions Transcriptional regulators like PLAGL2, EBF1, TCF7, KLF10 and SBNO2, previously not described in sepsis, are differentially expressed at early and late time points. Expression pattern for key biomarkers in this study is similar to that reported in human sepsis, indicating the suitability of this model for future studies of sepsis, and the observed differences in gene expression suggest species differences or differences in the response of blood leukocytes and peritoneal leukocytes

Role of an iron-dependent transcriptional regulator in the pathogenesis and host response to infection with Streptococcus pneumoniae.

Iron is a critical cofactor for many enzymes and is known to regulate gene expression in many bacterial pathogens. Streptococcus pneumoniae normally inhabits the upper respiratory mucosa but can also invade and replicate in lungs and blood. These anatomic sites vary considerably in both the quantity and form of available iron. The genome of serotype 4 pneumococcal strain TIGR4 encodes a putative iron-dependent transcriptional regulator (IDTR). A mutant deleted at idtr (?idtr) exhibited growth kinetics similar to parent strain TIGR4 in vitro and in mouse blood for up to 48 hours following infection. However, ?idtr was significantly attenuated in a murine model of sepsis. IDTR down-regulates the expression of ten characterized and putative virulence genes in nasopharyngeal colonization and pneumonia. The host cytokine response was significantly suppressed in sepsis with ?idtr. Since an exaggerated inflammatory response is associated with a poor prognosis in sepsis, the decreased inflammatory response could explain the increased survival with ?idtr. Our results suggest that IDTR, which is dispensable for pneumococcal growth in vitro, is associated with regulation of pneumococcal virulence in specific host environments. Additionally, IDTR ultimately modulates the host cytokine response and systemic inflammation that contributes to morbidity and mortality of invasive pneumococcal disease

Survival of mice infected with TIGR4 and Δ<i>idtr</i>.

<p>CBA/CaHN-Btk<i>^xid</i>/J mice were inoculated (A) intranasally with 10⁶ CFU and (B) intravenously with 10⁵ CFU of TIGR4 and Δ<i>idtr</i>. Kaplan Meier curves shown are a representative of triplicate experiments (n = 5 in each experiment).</p

Average bacterial counts from mouse blood TIGR4 and Δ<i>idtr</i>.

<p>A group of 5 mice each were infected intravenously with 10⁵ CFU of TIGR4 or Δ<i>idtr</i>. Blood samples at different time points were plated to determine bacterial counts. The error bars represent standard error of mean. ^**Significantly decreased as compared to TIGR4 infected blood counts (P<0.01).</p

Pneumococcal gene expression in Δ<i>idtr</i> in vitro and in vivo.

<p>Expression of ten pneumococcal genes in Δ<i>idtr</i> relative to TIGR4 in CDM (A) and from nasopharyngeal washes, lung homogenates and blood samples (B) was quantified by RT-PCR. Each experiment was performed using three separate biological sample, each done in triplicate.</p

Growth of TIGR4 and Δ<i>idtr</i> and Gram stain morphology of Δ<i>idtr</i> in vitro.

<p>The growth of TIGR4 and Δ<i>idtr</i> in CDM and iron depleted CDM was monitored by measuring absorbance at 600 nm. B) The morphology of Δ<i>idtr</i> was observed in (I) Iron depleted CDM (II) CDM by Gram staining. The results shown are average of three independent experiments cells grown in iron.</p

Schematic representation of Δ<i>idtr</i> construction.

<p>H-<i>HindIII</i>, B-<i>BamHI</i>. T1, T2 amplify the <i>tmp</i> cassette (495 bp); T1 and T2 have H and B at 5′ end. I1, I2 and I3, I4 amplify 5′ and 3′ end of <i>idtr</i>. I2 and I3 have H and B at 5′ end. I1, I2 amplify a 945 bp product and I3, I4 amplify a product of 489 bp.</p