Kirsten Sommer

Actin Re-Organization Induced by Chlamydia trachomatis Serovar D - Evidence for a Critical Role of the Effector Protein CT166 Targeting Rac

The intracellular bacterium Chlamydia trachomatis causes infections of urogenital tract, eyes or lungs. Alignment reveals homology of CT166, a putative effector protein of urogenital C. trachomatis serovars, with the N-terminal glucosyltransferase domain of clostridial glucosylating toxins (CGTs). CGTs contain an essential DXD-motif and mono-glucosylate GTP-binding proteins of the Rho/Ras families, the master regulators of the actin cytoskeleton. CT166 is preformed in elementary bodies of C. trachomatis D and is detected in the host-cell shortly after infection. Infection with high MOI of C. trachomatis serovar D containing the CT166 ORF induces actin re-organization resulting in cell rounding and a decreased cell diameter. A comparable phenotype was observed in HeLa cells treated with the Rho-GTPase-glucosylating Toxin B from Clostridium difficile (TcdB) or HeLa cells ectopically expressing CT166. CT166 with a mutated DXD-motif (CT166-mut) exhibited almost unchanged actin dynamics, suggesting that CT166-induced actin re-organization depends on the glucosyltransferase motif of CT166. The cytotoxic necrotizing factor 1 (CNF1) from E. coli deamidates and thereby activates Rho-GTPases and transiently protects them against TcdB-induced glucosylation. CNF1-treated cells were found to be protected from TcdB- and CT166-induced actin re-organization. CNF1 treatment as well as ectopic expression of non-glucosylable Rac1-G12V, but not RhoA-G14A, reverted CT166-induced actin re-organization, suggesting that CT166-induced actin re-organization depends on the glucosylation of Rac1. In accordance, over-expression of CT166-mut diminished TcdB induced cell rounding, suggesting shared substrates. Cell rounding induced by high MOI infection with C. trachomatis D was reduced in cells expressing CT166-mut or Rac1-G12V, and in CNF1 treated cells. These observations indicate that the cytopathic effect of C. trachomatis D is mediated by CT166 induced Rac1 glucosylation. Finally, chlamydial uptake was impaired in CT166 over-expressing cells. Our data strongly suggest CT166s participation as an effector protein during host-cell entry, ensuring a balanced uptake into host-cells by interfering with Rac-dependent cytoskeletal changes.

Systemic Inflammatory Response After Extremity or Truncal Fracture Operations

BACKGROUND The purpose of this study was to assess proinflammatory markers in blunt trauma patients regarding the relationship of these and blood loss and duration of surgery in different fracture locations. DESIGN Prospective, multicenter, nonrandomized cohort study. SETTING Three level I trauma centers. PATIENTS Sixty-eight blunt trauma patients, who did not require emergency operations and had sustained truncal or extremity fractures, were included. In two index patient groups, patients with spinal fractures (group SF, n = 24) and pelvic and acetabular fractures (group PAF, n = 21) underwent fixation of their fractures and were compared with a group of patients with isolated fractures (group FF, n = 28). Ten healthy volunteers served as controls. INTERVENTION Internal fixation of pelvic, acetabular and spinal fractures, intramedullary nailing of femoral fractures, measurement of proinflammatory cytokines. MAIN OUTCOME MEASURES From serially sampled central venous blood, the perioperative concentrations of interleukin-6 (IL-6) and IL-8 were evaluated during a 24-hour period and set into relation with the duration of surgery and the degree of blood loss. RESULTS Intramedullary instrumentation for isolated PAF caused a significant perioperative increase in the concentrations of IL-6 (preoperative: 16 pg/mL +/- 12 pg/mL, 7 hours: 89 pg/mL +/- 15 pg/mL, and 24 hours: 107 pg/mL +/- 27 pg/mL, p < 0.05). This increase was comparable with the isolated femoral fracture (group FF: IL-6 preoperative, 52 pg/mL +/- 12 pg/mL; 7 hours, 78 pg/mL +/- 14 pg/mL; and 24 hours, 120 pg/mL +/- 23 pg/mL, p = 0.02). The changes observed after spinal fracture fixations (group SF) were considerably lower (IL-6 preoperative: 11 pg/mL +/- 6 pg/mL, 7 hours: 16 pg/mL +/- 11 pg/mL, and 24 hours: 56 pg/mL +/- 19 pg/mL). The percent change of baseline IL-6 and IL-8 concentrations, and the blood loss in group PAF at 24 hours were positively correlated (IL-6 r = 0.72, p < 0.03, IL-8 0.67, p = 004) after insertion. No correlation with the duration of surgery was found. CONCLUSIONS The release of proinflammatory cytokines was higher in patients when their pelvic fractures were operated than in patients with spine fracture fixations, and was associated with the degree of blood loss. A higher increase in cytokine levels occurred when they were performed early (day 1-2) across all patient groups. The level of the released markers seems to be related to the magnitude of surgery, rather than to the duration of the procedure. This study supports the value of immunologic markers in determining subclinical changes during and after orthopedic surgical procedures.

A New Role of the Complement System: C3 Provides Protection in a Mouse Model of Lung Infection with Intracellular Chlamydia psittaci

The complement system modulates the intensity of innate and specific immunity. While it protects against infections by extracellular bacteria its role in infection with obligate intracellular bacteria, such as the avian and human pathogen Chlamydia (C.) psittaci, is still unknown. In the present study, knockout mice lacking C3 and thus all main complement effector functions were intranasally infected with C. psittaci strain DC15. Clinical parameters, lung histology, and cytokine levels were determined. A subset of infections was additionally performed with mice lacking C5 or C5a receptors. Complement activation occurred before symptoms of pneumonia appeared. Mice lacking C3 were ∼100 times more susceptible to the intracellular bacteria compared to wild-type mice, with all C3−/− mice succumbing to infection after day 9. At a low infective dose, C3−/− mice became severely ill after an even longer delay, the kinetics suggesting a so far unknown link of complement to the adaptive, protective immune response against chlamydiae. The lethal phenotype of C3−/− mice is not based on differences in the anti-chlamydial IgG response (which is slightly delayed) as demonstrated by serum transfer experiments. In addition, during the first week of infection, the absence of C3 was associated with partial protection characterized by reduced weight loss, better clinical score and lower bacterial burden, which might be explained by a different mechanism. Lack of complement functions downstream of C5 had little effect. This study demonstrates for the first time a strong and complex influence of complement effector functions, downstream of C3 and upstream of C5, on the outcome of an infection with intracellular bacteria, such as C. psittaci.

Identification of high- and low-virulent strains of Chlamydia pneumoniae by their characterization in a mouse pneumonia model

Contradicting reports exist about the pathogenicity of Chlamydia pneumoniae and the severity of the respiratory disease they cause. This study aimed to clarify, in mice, our hypothesis that marked differences in virulence of well-defined C. pneumoniae strains might exist for lung infections. C57BL/6J mice were intranasally infected with equal amounts of five different, identically prepared laboratory strains of C. pneumoniae. Based on the clinical score, weight, histopathological score, the granulocyte marker-enzyme myeloperoxidase, and the amount of Chlamydiae in the lung tissue, the C. pneumoniae isolates exhibited clear differences in overall growth characteristics or clearance, and pathological potential. Thus, we could identify chlamydial strains (Kajaani-K6 and CWL-029), where mice became seriously ill, as well as a relatively low-virulent isolate (TWAR-183). Cytokine profiles also varied drastically between the five strains in extent and kinetic. Our results indicate that C. pneumoniae isolates differ markedly with regard to their interaction with the host and their pathological potential. This might also be true for the infection in humans. Because the genomic diversity of C. pneumoniae is rather small, more subtle genomic deviations account most likely for the apparent functional differences. Our results will be useful to identify additional virulence factors in the future.

Severe pneumonia due to Parachlamydia acanthamoebae following intranasal inoculation: a mice model

Parachlamydia acanthamoebae is an obligate intracellular bacterium naturally infecting free-living amoebae. The role of this bacterium as an agent of pneumonia is suggested by sero-epidemiological studies and molecular surveys. Furthermore, P. acanthamoebae may escape macrophages microbicidal effectors. Recently, we demonstrated that intratracheal inoculation of P. acanthamoebae induced pneumonia in 100% of infected mice. However, the intratracheal route of infection is not the natural way of infection and we therefore developed an intranasal murine model. Mice inoculated with P. acanthamoebae by intranasal inoculation lost 18% of their weight up to 8 days post-inoculation. All mice presented histological signs of pneumonia at day 2, 4, 7, and 10 post-inoculation, whereas no control mice harboured signs of pneumonia. A 5-fold increase in bacterial load was observed from day 0 to day 4 post-inoculation. Lungs of inoculated mice were positive by Parachlamydia-specific immunohistochemistry 4 days post-inoculation, and P. acanthamoebae were localized within macrophages. Thus, we demonstrated that P. acanthamoebae induce a severe pneumonia in mice. This animal model (i) further supports the role of P. acanthamoebae as an agent of pneumonia, confirming the third Koch postulate, and (ii) identified alveolar macrophages as one of the initial cells where P. acanthamoebae is localized following infection.