RESUMEN
Burkholderia pseudomallei (Bp) is the causal agent of a high-morbidity/mortality disease syndrome known as melioidosis. This syndrome can range from acute fulminate disease to chronic, local, and disseminated infections that are often difficult to treat because Bp exhibits resistance to many antibiotics. Bp is a prime candidate for use in biologic warfare/terrorism and is classified as a Tier-1 select agent by HHS and APHIS. It is known that inbred mouse strains display a range of susceptibility to Bp and that the murine infection model is ideal for studying acute melioidosis. Here, we exploit a powerful mouse genetics resource that consists of a large family of BXD-type recombinant inbred strains, to perform genome-wide linkage analysis of the weight loss phenotype following pneumonic infection with Bp. We infected parental mice and 32 BXD strains with 50-100 CFU of Bp (strain 1026b) and monitored weight retention each day over an eleven-day time course. Using the computational tools in GeneNetwork, we performed genome-wide linkage analysis to identify an interval on chromosome 12 that appears to control the weight retention trait. We then analyzed and ranked positional candidate genes in this interval, several of which have intriguing connections with innate immunity, calcium homeostasis, lipid transport, host cell growth and development, and autophagy.
Asunto(s)
Infecciones por Burkholderia/patología , Neumonía/patología , Pérdida de Peso/genética , Animales , Burkholderia pseudomallei/aislamiento & purificación , Mapeo Cromosómico , Modelos Animales de Enfermedad , Femenino , Ligamiento Genético , Hibridación Genética , Masculino , Ratones EndogámicosRESUMEN
Working with infectious agents that require BSL-3 level containment agents offers many challenges for researchers. BSL-3 containment laboratories are usually not equipped with expensive specialty equipment that is needed for studies such as flow cytometric analysis, microscopy, and proteomic analyses. Therefore, for most researchers that are working with BSL-3 level infectious agents, removal of samples from BSL-3 laboratories for these types of studies is necessary, and methods for complete and dependable inactivation of the samples are required. In this report, we have carried out a thorough characterization of the effectiveness of paraformaldehyde fixation for inactivation of cell samples infected with the intracellular bacterial agents Burkholderia pseudomallei (Bp) and Francisella tularensis (Ft), both of which are Tier 1 select agent pathogens that require BSL-3 containment. We have demonstrated that cells infected with these pathogens are completely inactivated via 5-min treatment with 4% paraformaldehyde. Moreover, a 15-min treatment with 2% paraformaldehyde completely sterilized both Bp- and Ft-infected cells. These studies also revealed that Bp is significantly more sensitive to paraformaldehyde treatment than Ft. Our findings have clearly demonstrated that a 15-min treatment of Bp- or Ft-infected cells with 4% paraformaldehyde solution will allow for safe removal of the cell samples from BSL-3 laboratories for downstream studies.
Asunto(s)
Burkholderia pseudomallei/efectos de los fármacos , Burkholderia pseudomallei/fisiología , Contención de Riesgos Biológicos , Desinfección/métodos , Francisella tularensis/efectos de los fármacos , Francisella tularensis/fisiología , Viabilidad Microbiana/efectos de los fármacos , Animales , Investigación Biomédica/métodos , Células Cultivadas , Desinfectantes/farmacología , Formaldehído/farmacología , Ratones Endogámicos C57BL , Salud Laboral , Polímeros/farmacología , Factores de TiempoRESUMEN
BACKGROUND: A number of studies have revealed that Francisella tularensis (FT) suppresses innate immune responses such as chemokine/cytokine production and neutrophil recruitment in the lungs following pulmonary infection via an unidentified mechanism. The ability of FT to evade early innate immune responses could be a very important virulence mechanism for this highly infectious bacterial pathogen. RESULTS: Here we describe the characterization of a galU mutant strain of FT live vaccine strain (LVS). We show that the galU mutant was highly attenuated in a murine model of tularemia and elicited more robust innate immune responses than the wild-type (WT) strain. These studies document that the kinetics of chemokine expression and neutrophil recruitment into the lungs of mice challenged with the galU mutant strain are significantly more rapid than observed with WT FT, despite the fact that there were no observed differences in TLR2 or TLR4 signaling or replication/dissemination kinetics during the early stages of infection. We also show that the galU mutant had a hypercytotoxic phenotype and more rapidly induced the production of IL-1ß following infection either in vitro or in vivo, indicating that attenuation of the galU mutant strain may be due (in part) to more rapid activation of the inflammasome and/or earlier death of FT infected cells. Furthermore, we show that infection of mice with the galU mutant strain elicits protective immunity to subsequent challenge with WT FT. CONCLUSIONS: Disruption of the galU gene of FTLVS has little (if any) effect on in vivo infectivity, replication, or dissemination characteristics, but is highly attenuating for virulence. The attenuated phenotype of this mutant strain of FT appears to be related to its increased ability to induce innate inflammatory responsiveness, resulting in more rapid recruitment of neutrophils to the lungs following pneumonic infection, and/or to its ability to kill infected cells in an accelerated fashion. These results have identified two potentially important virulence mechanisms used by FT. These findings could also have implications for design of a live attenuated vaccine strain of FT because sublethal infection of mice with the galU mutant strain of FTLVS promoted development of protective immunity to WT FTLVS.