Antonina Torre

<p>BALB/c mice were immunized once i.m. with 4C-Staph/T7-alum or 4C-Staph/alum. Control mice were injected with T7-alum or alum alone. After 12 days, mice were challenged with <i>S</i>. <i>aureus</i> Newman strain. (<b>A</b>) Kidney abscess model. Mice (n = 28–32) were injected i.v. with 2 x 10<sup>7</sup> CFU. Four days later, both kidneys of each mouse were homogenized in pool and CFU enumerated. Each symbol represents one mouse, and data are the merge of three independent experiments. Mean ± SEM of each group are shown. The dotted line indicates the lower limit of detection (LLD). *<i>p</i> < 0.05, **<i>p</i> < 0.01 by one-way ANOVA and Sidak's multiple comparisons test. Number of survivors with non-detectable CFU (CFU ND) in kidneys/total number of survivors and corresponding percentages are reported above the graph. (<b>B-C</b>) Peritonitis model. Mice (n = 32) were injected i.p. with 5 x 10<sup>8</sup> CFU. Survival was monitored for 30 days after challenge. Data are the merge of three independent experiments. ***<i>p</i> < 0.001 by Log-rank test. (<b>C</b>) Thirty days after <i>S</i>. <i>aureus</i> infection, survivors were euthanized, both kidneys were homogenized and CFU enumerated. Each symbol represents one mouse. Mean ± SEM of each group is shown. **<i>p</i> < 0.01 by unpaired Student <i>t</i> test, one-tailed. Number of survivors with CFU ND in kidneys/total number of survivors and corresponding percentages are reported above the graph.</p

Staphylococcus aureusalpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane causing lysis, apoptosis, and junction disruption. Herein we present the design of a newly engineeredS. aureusalpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata and its structure, obtained by transmission electron microscopy and single particle reconstruction analysis, resembles the cap of the wild type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 as well as to lack both haemolytic and cytotoxic activity. Immunological studies using human sera as well as sera of mice convalescent fromS. aureusinfection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective i...

In vivo imaging of bioluminescent bacteria permits their visualization in infected mice, allowing spatial and temporal evaluation of infection progression. Most available bioluminescent strains were obtained by integration of the luciferase genes into the bacterial chromosome, a challenging and time-consuming approach. Recently, episomal plasmids were used, which were introduced in bacteria and expressed all genes required for bioluminescence emission. However, the plasmid was progressively lost in vitro and in vivo, if bacteria were not maintained under antibiotic selective pressure. Increased stability could be obtained inserting into the plasmid backbone sequences that assured plasmid partition between daughter bacterial cells, or caused death of bacteria that had lost the plasmid. So far, no detailed analysis was performed of either plasmid stability in vivo or contribution of different stabilizing sequence types. Here we report the construction of a plasmid, which includes the Photorhabdus luminescens lux cassette expressed under the control of a Staphylococcus aureus specific gene promoter, and toxin/antitoxin (T/A) and partition sequences (Par) conferring stability and transmissibility of the plasmid. Following infection of mice with S. aureus carrying this plasmid, we demonstrated that the promoter-lux fusion was functional in vivo, that the plasmid was retained by 70-100 % of bacterial cells 7 days post-infection, and that both stabilizing sequence types were required to maximize plasmid retention. These data suggest that the plasmid can be a valuable tool to study gene expression and bacterial spread in small laboratory animals infected with S. aureus or possibly other Gram-positive human pathogens.