Mitogen-activated protein kinases (MAPKs) are modulated during in vitro and in vivo infection with the intracellular bacterium Burkholderia pseudomallei.

Burkholderia pseudomallei is a Gram-negative intracellular bacterium that causes the disease melioidosis. The disease can be fatal if left untreated or when antibiotic therapy is delayed and total clearance of the pathogen from the host is often not accomplished with current therapies. Thus, new therapeutic approaches for the treatment of infections caused by B. pseudomallei are required. To better understand host responses to B. pseudomallei infection, the activation of key proteins involved in the TLR inflammatory cascade was measured by western blotting. Activation of the mitogen-activated protein kinases (MAPKs) p38 and ERK were both significantly altered during both in vitro and in vivo infection. In considering an approach for therapy of B. pseudomallei infection the inhibition of ERK was achieved in vitro using the inhibitor PD0325901, along with decreased TNF-α production. However, the reduction in phosphorylated ERK and TNF-α release did not correspond with decreased bacterial replication or enhance clearance from infected macrophages. Despite this apparent lack of effect on the intracellular growth of B. pseudomallei in vitro, it is not clear what effect inhibition of ERK activation might have on outcome of disease in vivo. It may be that decreasing the levels of TNF-α in vivo could aid in reducing the overactive immune response that is known to ensue following B. pseudomallei infection, thereby increasing host survival.

Mitogen-activated protein kinases (MAPKs) are modulated during Francisella tularensis infection, but inhibition of extracellular-signal-regulated kinases (ERKs) is of limited therapeutic benefit.

Francisella tularensis is a Gram-negative intracellular bacterium that causes the disease tularemia. The disease can be fatal if left untreated and there is currently no licenced vaccine available; the identification of new therapeutic targets is therefore required. Toll-like receptors represent an interesting target for therapeutic modulation due to their essential role in generating immune responses. In this study, we analysed the in vitro expression of the key mitogen-activated protein kinases (MAPKs) p38, JNK and ERK in murine alveolar macrophages during infection with F. tularensis. The phosphorylation profile of ERK highlighted its potential as a target for therapeutic modulation and subsequently the effect of ERK manipulation was measured in a lethal intranasal F. tularensis in vivo model of infection. The selective ERK1/2 inhibitor PD0325901 was administered orally to mice either pre- or post-challenge with F. tularensis strain LVS. Both treatment regimens selectively reduced ERK expression, but only the pre-exposure treatment produced decreased bacterial burden in the spleen and liver, which correlated with a significant reduction in the pro-inflammatory cytokines IFN-γ, MCP-1, IL-6, and TNF-α. However, no overall improvements in survival were observed for treated animals in this study. ERK may represent a useful therapeutic target where selective dampening of the immune response (to control the damaging pathology seen during infection) is combined with antibiotic treatment required to eradicate bacterial infection. This combination treatment strategy has been shown to be effective in other models of tularemia.

Construction of an inducible system for the analysis of essential genes in Yersinia pestis.

Yersinia pestis, a Gram negative bacterium, causes bubonic and pneumonic plague. Emerging antibiotic resistance in clinical isolates is driving a need to develop novel antibiotics to treat infection by this transmissible and highly virulent pathogen. Proteins required for viability, so called essential genes, are attractive potential therapeutic targets, however, confirmation of essentiality is problematic. For the first time, we report the development of a system that allows the rapid determination of Y. pestis gene essentiality through mutagenesis and inducible expression of a plasmid borne copy of the target gene. Using this approach, we have confirmed the uridine monophosphate kinase PyrH as an essential protein in Y. pestis. This methodology and the tools we have developed will allow the confirmation of other putative essential genes in this dangerous pathogen, and facilitate the identification of novel targets for antimicrobial development.