In vivo analysis of staphylococcus aureus-infected mice reveals differential temporal and spatial expression patterns of fhuD2

Staphylococcus aureus is an opportunistic human pathogen and a major cause of invasive infections such as bacteremia, endocarditis, pneumonia and wound infections. FhuD2 is a staphylococcal lipoprotein involved in the uptake of iron-hydroxymate and is under the control of the iron uptake regulator Fur. The protein is part of an investigational multi-component vaccine formulation that has shown protective efficacy in several murine models of infection. Even though fhuD2 expression was shown to be upregulated in murine kidneys infected with S. aureus, it is unknown whether the bacterium undergoes increased iron deprivation during prolonged infection. Furthermore, different infection niches of S. aureus might provide different environments and iron availability resulting in different fhuD2 expression pattern within different host organs. To address these questions, we characterized the in vitro expression of the fhuD2 gene and confirmed Fur-dependent iron-regulation of its expression. We further investigated its expression in mice infected with a bioluminescent reporter strain of S. aureus expressing the luciferase operon under the control of the fhuD2 promoter. The emission of bioluminescence in different organs was followed over a seven-day time course, as well as quantitative real-time PCR analysis of the RNA transcribed from the endogenous fhuD2 gene. Using this approach, we could show that fhuD2 expression was induced during infection in all organs analyzed and that differences in expression were observed in the temporal expression profiles, and between infected organs. Our data suggest that S. aureus undergoes increased iron deprivation during progression of infection in diverse host organs and accordingly induces dedicated iron acquisition mechanisms. Since FhuD2 plays a central role in providing the pathogen with the required iron, further knowledge of the patterns of fhuD2 expression in vivo during infection is instrumental in better defining the role of this antigen in S. aureus pathogenesis and as a vaccine antigen

A stable luciferase reporter plasmid for in vivo imaging in murine models of Staphylococcus aureus infections

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

<i>In vivo</i> analysis of <i>staphylococcus aureus</i>-infected mice reveals differential temporal and spatial expression patterns of fhud2

Staphylococcus aureus is an opportunistic human pathogen and a major cause of invasive infections such as bacteremia, endocarditis, pneumonia, and wound infections. FhuD2 is a staphylococcal lipoprotein involved in the uptake of iron-hydroxymate and is under the control of the iron uptake regulator Fur. This protein is part of an investigational multicomponent vaccine formulation that has shown protective efficacy in several murine models of infection. Even though fhuD2 expression has been shown to be upregulated in murine kidneys infected with S. aureus, it is not known whether the bacterium undergoes increased iron deprivation during prolonged infection. Furthermore, different S. aureus infection niches might provide different environments and levels of iron availability, resulting in different fhuD2 expression patterns among organs of the same host. To address these questions, we characterized the in vitro expression of the fhuD2 gene and confirmed Fur-dependent regulation of its expression. We further investigated its expression in mice infected with a bioluminescent reporter strain of S. aureus expressing the luciferase operon under the control of the fhuD2 promoter. The emission of bioluminescence in different organs was followed over a 7-day time course, and quantitative real-time PCR analysis of the RNA transcribed from the endogenous fhuD2 gene was performed. Using this approach, we were able to show that fhuD2 expression was induced during infection in all organs analyzed and that differences in expression were observed at different time points and in different infected organs. Our data suggest that S. aureus undergoes increased iron deprivation during the progression of infection in diverse host organs and accordingly induces dedicated iron acquisition mechanisms. Since FhuD2 plays a central role in providing the pathogen with the required iron, further knowledge of the patterns of fhuD2 expression in vivo during infection will be instrumental in better defining the role of this antigen in S. aureus pathogenesis and as a vaccine antigen

<i>In vivo</i> analysis of <i>staphylococcus aureus</i>-infected mice reveals differential temporal and spatial expression patterns of fhud2

Staphylococcus aureus is an opportunistic human pathogen and a major cause of invasive infections such as bacteremia, endocarditis, pneumonia, and wound infections. FhuD2 is a staphylococcal lipoprotein involved in the uptake of iron-hydroxymate and is under the control of the iron uptake regulator Fur. This protein is part of an investigational multicomponent vaccine formulation that has shown protective efficacy in several murine models of infection. Even though fhuD2 expression has been shown to be upregulated in murine kidneys infected with S. aureus, it is not known whether the bacterium undergoes increased iron deprivation during prolonged infection. Furthermore, different S. aureus infection niches might provide different environments and levels of iron availability, resulting in different fhuD2 expression patterns among organs of the same host. To address these questions, we characterized the in vitro expression of the fhuD2 gene and confirmed Fur-dependent regulation of its expression. We further investigated its expression in mice infected with a bioluminescent reporter strain of S. aureus expressing the luciferase operon under the control of the fhuD2 promoter. The emission of bioluminescence in different organs was followed over a 7-day time course, and quantitative real-time PCR analysis of the RNA transcribed from the endogenous fhuD2 gene was performed. Using this approach, we were able to show that fhuD2 expression was induced during infection in all organs analyzed and that differences in expression were observed at different time points and in different infected organs. Our data suggest that S. aureus undergoes increased iron deprivation during the progression of infection in diverse host organs and accordingly induces dedicated iron acquisition mechanisms. Since FhuD2 plays a central role in providing the pathogen with the required iron, further knowledge of the patterns of fhuD2 expression in vivo during infection will be instrumental in better defining the role of this antigen in S. aureus pathogenesis and as a vaccine antigen

A stable luciferase reporter plasmid for in vivo imaging in murine models of Staphylococcus aureus infections

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.</p

One dose of 4C-Staph/T7-alum induces functional antibodies.

<p>BALB/c mice (n = 16) were immunized once with 4C-Staph/T7-alum or 4C-Staph/alum. Control mice were injected with T7-alum or alum alone. (<b>A</b>) Vaccine-specific serum IgG titers measured 12 (d12) and 32 (d32) days after vaccination. IgG concentrations in control sera (open symbols) are reported only for d32. Each symbol represents one mouse, and data are the merge of two independent experiments. Median with interquartile range of each group is also shown. *<i>p</i> < 0.05, ****<i>p</i> < 0.0001 by Kruskal-Wallis test and Dunn's multiple comparisons test. (<b>B</b>) Hla neutralizing activity of pooled sera from vaccinated mice (n = 16, same animals as in A) was assessed on rabbit RBCs and expressed as effective dilution that neutralized 50% of Hla lytic activity (ED₅₀). No hemolysis inhibition was detected (ED₅₀ < 6) in pre-immune sera or in sera from adjuvant-treated mice. Lack of overlap in the 95% confidence intervals between the ED₅₀ of sera from mice vaccinated with 4C-Staph/alum (30.3 to 51.4) vs. 4C-Staph/T7-alum (114.6 to 318.7) by 32 days indicates a difference significant with <i>p</i> < 0.05. Bars represent SEM. (<b>C</b>) Vaccine-specific IgG1 and IgG2a. Columns represent median MFI with interquartile range of pooled sera from vaccinated mice (n = 16, same pools as in B) bled at d32. **<i>p</i> < 0.01, ***<i>p</i> < 0.001 by unpaired Student <i>t</i> test, two-tailed. (<b>D</b>) Hla_H35L-specific IgM. Columns represent median MFI with interquartile range of sera from vaccinated mice (n = 12) bled at d12. IgM specific for EsxAB, FhuD2 and Csa1A were at the limit of detection (data not shown). Data shown are the merge of two independent experiments.</p

One dose of 4C-Staph/T7-alum induces protective antibodies.

<p>Sera from mice immunized with 4C-Staph/T7-alum (immune serum), or T7-alum as negative control (control serum), by 32 days were pooled and injected i.v. (150 μl/mouse) in naïve BALB/c mice (n = 16) 24 h before i.p. challenge with <i>S</i>. <i>aureus</i>. <b>(A</b>) Survival was monitored for 15 days post challenge. Data are the merge of two independent experiments. ***<i>p</i> < 0.001 by Log-rank test. <b>(B</b>) Fifteen days after <i>S</i>. <i>aureus</i> infection, survivors were euthanized, both kidneys were homogenized and CFU enumerated. Each symbol represents one mouse. <b>(C</b>) B cell/antibody-deficient J_H mice, or BALB/c (wt) as control, were immunized with 4C-Staph/T7-alum or T7-alum alone. Twelve days after vaccination, mice (n = 25 for 4C-Staph/T7-alum; n = 15 for T7-alum) were challenged i.p. with <i>S</i>. <i>aureus</i> and their survival was monitored for 15 days. Data are the merge of four independent experiments. ***<i>p</i> < 0.001 by Log-rank test.</p

One Dose of <i>Staphylococcus aureus</i> 4C-Staph Vaccine Formulated with a Novel TLR7-Dependent Adjuvant Rapidly Protects Mice through Antibodies, Effector CD4⁺ T Cells, and IL-17A

<div><p>A rapidly acting, single dose vaccine against <i>Staphylococcus aureus</i> would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent <i>S</i>. <i>aureus</i> candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4⁺ T cells not only reduced survival but also increased <i>S</i>. <i>aureus</i> load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by <i>in vivo</i> neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against <i>S</i>. <i>aureus</i> through the combined actions of antibodies, CD4⁺ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.</p></div

One dose of 4C-Staph/T7-alum vaccine protects better than 4C-Staph/alum in kidney abscess and peritonitis models of <i>S</i>. <i>aureus</i> infection.

<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 10⁷ 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 10⁸ 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

IL-17A neutralization in mice vaccinated with 4C-Staph/T7-alum has no effect on survival but increases the bacterial load in kidneys upon i.p. <i>S</i>. <i>aureus</i> challenge.

<p>Twelve days after vaccination with 4C-Staph/T7-alum or T7-alum, BALB/c mice (n = 15–16) were challenged i.p. with <i>S</i>. <i>aureus</i>. Mice were injected i.p. with neutralizing mAb (neutr. Ab): (<b>A</b>) anti-IL-17A; (<b>B</b>) anti-IFN-γ; or (<b>C</b>) anti-IL-17A and anti-IFN-γ 3 h before challenge and every other day for 15 days after challenge. Control mice were injected with isot. ctr. (<b>A-C</b>, left panels) Survival was monitored for 15 days after challenge. Data are the merge of two independent experiments. No statistically significant differences between mice vaccinated with 4C-Staph/T7-alum treated with neutr. Ab or isot. ctr. (Log-rank test). (<b>A-C</b>, right panels) Fifteen days after <i>S</i>. <i>aureus</i> inoculation, survivors (n = 7–8) were euthanized, both kidneys homogenized and CFU enumerated. Each symbol represents a mouse. Mean ± SEM. One representative experiment out of two is shown. The dotted line indicates the lower limit of CFU detection. *<i>p</i> < 0.01; ***<i>p</i> < 0.001 by unpaired Student <i>t</i> test, one-tailed.</p