Effective, Broad Spectrum Control of Virulent Bacterial Infections Using Cationic DNA Liposome Complexes Combined with Bacterial Antigens

Protection against virulent pathogens that cause acute, fatal disease is often hampered by development of microbial resistance to traditional chemotherapeutics. Further, most successful pathogens possess an array of immune evasion strategies to avoid detection and elimination by the host. Development of novel, immunomodulatory prophylaxes that target the host immune system, rather than the invading microbe, could serve as effective alternatives to traditional chemotherapies. Here we describe the development and mechanism of a novel pan-anti-bacterial prophylaxis. Using cationic liposome non-coding DNA complexes (CLDC) mixed with crude F. tularensis membrane protein fractions (MPF), we demonstrate control of virulent F. tularensis infection in vitro and in vivo. CLDC+MPF inhibited bacterial replication in primary human and murine macrophages in vitro. Control of infection in macrophages was mediated by both reactive nitrogen species (RNS) and reactive oxygen species (ROS) in mouse cells, and ROS in human cells. Importantly, mice treated with CLDC+MPF 3 days prior to challenge survived lethal intranasal infection with virulent F. tularensis. Similarly to in vitro observations, in vivo protection was dependent on the presence of RNS and ROS. Lastly, CLDC+MPF was also effective at controlling infections with Yersinia pestis, Burkholderia pseudomallei and Brucella abortus. Thus, CLDC+MPF represents a novel prophylaxis to protect against multiple, highly virulent pathogens

Role for the Burkholderia pseudomallei Capsular Polysaccharide Encoded by the wcb Operon in Acute Disseminated Melioidosis▿†

The capsular polysaccharide of Burkholderia pseudomallei is an essential virulence determinant that is required for protection from host serum cidal activity and opsonophagocytosis. In this study, the immune response directed against a B. pseudomallei capsule mutant (JW270) was investigated in an acute respiratory murine model. JW270 was significantly attenuated in this model (∼2 logs) to levels resembling those of avirulent Burkholderia thailandensis. At lethal doses, JW270 colonized the lung, liver, and spleen at levels similar to the wild-type strain levels and was found to trigger reduced pathology in the liver and spleen. Several cytokine responses were altered in these tissues, and importantly, the levels of gamma interferon were reduced in the livers and spleens of JW270-infected mice but not in the lungs. These results suggest that the capsular polysaccharide of B. pseudomallei is a critical virulence determinant in respiratory tract infections and that it is an important antigen for generating the Th1 immune response commonly observed in systemic melioidosis. Furthermore, the data suggest that host recognition of B. pseudomallei capsular polysaccharide in the lungs may not be as important to the disease outcome as the innate immune response in the peripheral organs

Type 3 secretion system cluster 3 is a critical virulence determinant for lung-specific melioidosis.

Burkholderia pseudomallei, the bacterial agent of melioidosis, causes disease through inhalation of infectious particles, and is classified as a Tier 1 Select Agent. Optical diagnostic imaging has demonstrated that murine respiratory disease models are subject to significant upper respiratory tract (URT) colonization. Because human melioidosis is not associated with URT colonization as a prominent presentation, we hypothesized that lung-specific delivery of B. pseudomallei may enhance our ability to study respiratory melioidosis in mice. We compared intranasal and intubation-mediated intratracheal (IMIT) instillation of bacteria and found that the absence of URT colonization correlates with an increased bacterial pneumonia and systemic disease progression. Comparison of the LD50 of luminescent B. pseudomallei strain, JW280, in intranasal and IMIT challenges of albino C57BL/6J mice identified a significant decrease in the LD50 using IMIT. We subsequently examined the LD50 of both capsular polysaccharide and Type 3 Secretion System cluster 3 (T3SS3) mutants by IMIT challenge of mice and found that the capsule mutant was attenuated 6.8 fold, while the T3SS3 mutant was attenuated 290 fold, demonstrating that T3SS3 is critical to respiratory melioidosis. Our previously reported intranasal challenge studies, which involve significant URT colonization, did not identify a dissemination defect for capsule mutants; however, we now report that capsule mutants exhibit significantly reduced dissemination from the lung following lung-specific instillation, suggesting that capsule mutants are competent to spread from the URT, but not the lung. We also report that a T3SS3 mutant is defective for dissemination following lung-specific delivery, and also exhibits in vivo growth defects in the lung. These findings highlight the T3SS3 as a critical virulence system for respiratory melioidosis, not only in the lung, but also for subsequent spread beyond the lung using a model system uniquely capable to characterize the fate of lung-delivered pathogen

Bacterial enumeration at moribund disease for respiratory melioidosis models.

<p>Groups of 5 female albino C57BL/6J mice were infected by either the i.n. (10^5.1 CFU) or IMIT (10^4.6 CFU) routes of infection and euthanized at moribund disease endpoints. Bacteria were enumerated from tissues homogenized in 1 ml PBS, from a 1 ml PBS BAL collection, or from cardiac-drawn blood. Bacterial burden was calculated as CFU/tissue (lung, liver, and spleen) or bacteria per ml of body fluid (BAL and blood). Significant differences between log transformed data were evaluated by 2-way ANOVA with Bonferroni multiple comparisons (n.s., not significant; *, p<0.05; **, p<0.01; ****, p<0.0001).</p

Detection of pulmonary growth rates of <i>B. pseudomallei</i> mutants <i>in vivo</i>.

<p>Groups of 5 female albino C57BL/6J mice were infected with wild type JW280 (10^4.2 CFU), Δ<i>wcb</i> capsule mutant (10^6.0 CFU), or Δ<i>sctU</i>Bp3 T3SS3 mutant (10^6.5 CFU), and monitored by optical diagnostic imaging once to twice daily. ROIs from the dorsally-imaged thoracic cavity were plotted as a function of infection time for each mutant. The 95% LOD was calculated for the background luminescence of uninfected mice and indicated as a dotted horizontal line. The calculated doubling rate of bioluminescent signal of each strain is indicated.</p

Host response to capsule and T3SS3 mutants of <i>B. pseudomallei</i>.

<p>Groups of 5 female albino C57BL/6J mice were challenged by the IMIT route of infection with increasing doses of either Δ<i>wcb</i> capsule mutant (A) or Δ<i>sctU</i>_Bp3 T3SS3 mutant (B) in the luminescent <i>B. pseudomallei</i> strain JW280 background. Mice were monitored for 14 days (336 hr) for disease progression and euthanized at the onset of moribund disease presentation. Survival curves are used to present the dose-dependent response of the host to increasing bacterial challenges of <i>B. pseudomallei</i> mutants. The MTTD was calculated for groups with ≥50% mortality, as indicated.</p

Detection of dissemination of <i>B. pseudomallei</i> mutants by optical imaging.

<p>Groups of 5 female albino C57BL/6J mice were challenged by the IMIT route of infection with either wild type luminescent <i>B. pseudomallei</i> strain JW280, the Δ<i>wcb</i> capsule mutant, or the Δ<i>sctU</i>_Bp3 T3SS3 mutant. Representative images of disease endpoints are presented, with uniform image settings adjusted to a range of 2.5×10³ to 3×10⁴ p/s/cm2/sr on a logarithmic scale.</p

Bacterial enumeration of <i>B. pseudomallei</i> mutants in moribund respiratory disease.

<p>Groups of 5 female albino C57BL/6J mice were infected with wild type JW280 (10^4.2 CFU), Δ<i>wcb</i> capsule mutant (10^6.0 CFU), or Δ<i>sctU</i>Bp3 T3SS3 mutant (10^6.5 CFU), and euthanized at moribund disease endpoints. Bacteria were enumerated from tissues <i>ex vivo</i> by optical imaging (left Y-axis: cps/tissue) with presentation of the estimated tissue CFU burdens based on calculated tissue-specific cps:CFU correlation (right Y-axis: CFU/tissue est.) for lung (A), liver (B) and spleen (C). The 95% LOD was calculated as a technical background luminescence and indicated as a dotted horizontal line. Data points below the 95% LOD were set to the 95% LOD value. Significant differences (1-way ANOVA with Tukey posttest) between log transformed data sets are indicated with an adjoining line (*, p<0.05; **, p<0.01; ***, p<0.001).</p