Yersinia pestis Endowed with Increased Cytotoxicity Is Avirulent in a Bubonic Plague Model and Induces Rapid Protection against Pneumonic Plague

An important virulence strategy evolved by bacterial pathogens to overcome host defenses is the modulation of host cell death. Previous observations have indicated that Yersinia pestis, the causative agent of plague disease, exhibits restricted capacity to induce cell death in macrophages due to ineffective translocation of the type III secretion effector YopJ, as opposed to the readily translocated YopP, the YopJ homologue of the enteropathogen Yersinia enterocolitica O∶8. This led us to suggest that reduced cytotoxic potency may allow pathogen propagation within a shielded niche, leading to increased virulence. To test the relationship between cytotoxic potential and virulence, we replaced Y. pestis YopJ with YopP. The YopP-expressing Y. pestis strain exhibited high cytotoxic activity against macrophages in vitro. Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 107-fold reduction in virulence. Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain. The subcutaneous administration of the cytotoxic Y. pestis strain appears to activate a rapid and potent systemic, CTL-independent, immunoprotective response, allowing the organism to overcome simultaneous coinfection with 10,000 LD50 of virulent Y. pestis. Moreover, three days after subcutaneous administration of this strain, animals were also protected against septicemic or primary pneumonic plague. Our findings indicate that an inverse relationship exists between the cytotoxic potential of Y. pestis and its virulence following subcutaneous infection. This appears to be associated with the ability of the engineered cytotoxic Y. pestis strain to induce very rapid, effective and long-lasting protection against bubonic and pneumonic plague. These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed new light on the virulence strategies of Y. pestis in nature

Economic Assessment of an Integrated Membrane System for Secondary Effluent Polishing for Unrestricted Reuse

Extra treatment stages are required to polish the secondary effluent for unrestricted reuse, primarily for agricultural irrigation. Improved technology for the removal of particles, turbidity, bacteria and cysts, without the use of disinfectants is based on MicroFiltration (MF) and UltraFiltration (UF) membrane technology and in series with Reverse Osmosis (RO) for dissolved solids removal. Field experiments were conducted using a mobile UF and RO membrane pilot unit at a capacity of around 1.0 m3/hr. A management model was defined and tested towards optimal polishing of secondary effluent. The two major purposes of the management model are: (i) to delineate a methodology for economic assessment of optimal membrane technology implementation for secondary effluent upgrading for unrestricted use, and; (ii) to provide guidelines for optimal RO membrane selection in regards to the pretreatment stage. The defined linear model takes into account the costs of the feed secondary effluent, the UF pretreatment and the RO process. Technological constraints refer primarily to the longevity of the membrane and their performance. Final treatment cost (the objective function) includes investment, operation and maintenance expenses, UF pretreatment, RO treatment, post treatment and incentive for low salinity permeate use. The cost range of water for irrigation according to the model is between 15 and 42 US cents per m3

YopP-expressing variant of Y. pestis activates a potent innate immune response affording cross-protection against yersiniosis and tularemia [corrected].

Plague, initiated by Yersinia pestis infection, is a rapidly progressing disease with a high mortality rate if not quickly treated. The existence of antibiotic-resistant Y. pestis strains emphasizes the need for the development of novel countermeasures against plague. We previously reported the generation of a recombinant Y. pestis strain (Kim53ΔJ+P) that over-expresses Y. enterocolitica YopP. When this strain was administered subcutaneously to mice, it elicited a fast and effective protective immune response in models of bubonic, pneumonic and septicemic plague. In the present study, we further characterized the immune response induced by the Kim53ΔJ+P recombinant strain. Using a panel of mouse strains defective in specific immune functions, we observed the induction of a prompt protective innate immune response that was interferon-γ dependent. Moreover, inoculation of mice with Y. pestis Kim53ΔJ+P elicited a rapid protective response against secondary infection by other bacterial pathogens, including the enteropathogen Y. enterocolitica and the respiratory pathogen Francisella tularensis. Thus, the development of new therapies to enhance the innate immune response may provide an initial critical delay in disease progression following the exposure to highly virulent bacterial pathogens, extending the time window for successful treatment

Kim53ΔJ+P is highly virulent following systemic or airway infection.

<p>(A) Systemic infection (i.v. inoculation). Groups of 6 mice were infected intravenously with a dose of 2×10³ cfu of Kim53pGFP (circle) or Kim53ΔJ+P (triangle), and mortality was monitored daily for 14 days. (B) Airway infection (i.n. inoculation). Groups of 5 mice were infected intranasally with 6×10⁴ cfu of Kim53pGFP (circle) or Kim53ΔJ+P (triangle). (C) Dissemination of <i>Y. pestis</i> strains to blood and internal organs following intranasal infection. Groups of 5 mice were infected intranasally with 2×10⁵ cfu of Kim53pGFP (black symbol) or Kim53ΔJ+P (white symbol) and sacrificed 48 hours post-infection. Bacterial concentration in blood and total bacterial loads in lungs, mediastinal lymph nodes (MSLNs) and the spleen were determined as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005938#pone-0005938-g002" target="_blank">Figure 2</a>.</p

Kim53ΔJ+P is deficient in dissemination to target organs and to the blood following subcutaneous infection.

<p>Mice were infected subcutaneously with 1×10⁴ cfu of either Kim53pGFP (black symbols) or Kim53ΔJ+P (white symbols). Animals (4–5 per group) were sacrificed at 60 hours post-infection. Blood was collected, and the draining inguinal lymph nodes (ILN) and spleens were harvested, homogenized in 1 ml PBS and cultured on brain heart infusion agar plates at 28°C for 48 hrs. Values in Figure represent total bacterial loads in infected organs (cfu/organ), or bacterial concentration in blood (cfu/ml). LOD, limit of detection. Horizontal bars represent the average value of bacterial load in each case. Differences in bacterial concentrations in blood and internal organs were analyzed by the non-parametric Mann-Whitney test.</p

<i>Y. pestis</i> Kim53ΔJ+P strain confers prolonged protection against plague.

<p>(A) Mice (7–8 per group) were infected subcutaneously with 1×10⁵ cfu of Kim53ΔJ+P (circle); EV76 (× symbol); or EV76ΔJ+P (triangle). Control animals were treated with saline (square). Animals were bled for ELISA determination of anti-F1 antibody titers 45 days following immunization. Titers following immunization with Kim53ΔJ+P ranged from 64,000 to 100,000, with GMT of 18,500, while titers for the EV76 or EV76ΔJ+P immunized animals ranged from 640 to less than 10, with GMT of 30 and 10, respectively. Animals were challenged subcutaneously with 1×10³ cfu (∼1×10³ LD₅₀) of <i>Y. pestis</i> Kim53 (panel A) or intranasally with 8×10³ cfu (∼15 LD₅₀) of <i>Y. pestis</i> Kim53 (panel B).</p