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A Burkholderia pseudomallei macrophage infectivity potentiator-like protein has rapamycin-inhibitable peptidylprolyl isomerase activity and pleiotropic effects on virulence.

ABSTRACT: Macrophage infectivity potentiators (Mips) are a group of virulence factors encoded by pathogenic bacteria such as Legionella, Chlamydia, and Neisseria species. Mips are part of the FK506-binding protein (FKBP) family, whose members typically exhibit peptidylprolyl cis-trans isomerase (PPIase) activity which is inhibitable by the immunosuppressants FK506 and rapamycin. Here we describe the identification and characterization of BPSS1823, a Mip-like protein in the intracellular pathogen Burkholderia pseudomallei. Recombinant BPSS1823 protein has rapamycin-inhibitable PPIase activity, indicating that it is a functional FKBP. A mutant strain generated by deletion of BPSS1823 in B. pseudomallei exhibited a reduced ability to survive within cells and significant attenuation in vivo, suggesting that BPSS1823 is important for B. pseudomallei virulence. In addition, pleiotropic effects were observed with a reduction in virulence mechanisms, including resistance to host killing mechanisms, swarming motility, and protease production.

SUBMITTER: Norville IH

PROVIDER: S-EPMC3257933 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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A Burkholderia pseudomallei macrophage infectivity potentiator-like protein has rapamycin-inhibitable peptidylprolyl isomerase activity and pleiotropic effects on virulence.

Norville Isobel H IH Harmer Nicholas J NJ Harding Sarah V SV Fischer Gunter G Keith Karen E KE Brown Katherine A KA Sarkar-Tyson Mitali M Titball Richard W RW

Infection and immunity 20110822 11

Macrophage infectivity potentiators (Mips) are a group of virulence factors encoded by pathogenic bacteria such as Legionella, Chlamydia, and Neisseria species. Mips are part of the FK506-binding protein (FKBP) family, whose members typically exhibit peptidylprolyl cis-trans isomerase (PPIase) activity which is inhibitable by the immunosuppressants FK506 and rapamycin. Here we describe the identification and characterization of BPSS1823, a Mip-like protein in the intracellular pathogen Burkholde ...[more]

PMID: 21859853

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Project description:IntroductionMelioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a disease endemic in many tropical countries globally. Clinical presentation is highly variable, ranging from asymptomatic to fatal septicemia, and thus the outcome of infection can depend on the host immune responses. The aims of this study were to firstly, characterize the macrophage immune response to B. pseudomallei and secondly, to determine whether the immune response was modified in the presence of novel inhibitors targeting the virulence factor, the macrophage infectivity potentiator (Mip) protein. We hypothesized that inhibition of Mip in B. pseudomallei would disarm the bacteria and result in a host beneficial immune response.MethodsMurine macrophage J774A.1 cells were infected with B. pseudomallei K96243 in the presence of small-molecule inhibitors targeting the Mip protein. RNA-sequencing was performed on infected cells four hours post-infection. Secreted cytokines and lactose dehydrogenase were measured in cell culture supernatants 24 hours post-infection. Viable, intracellular B. pseudomallei in macrophages were also enumerated 24 hours post-infection.ResultsGlobal transcriptional profiling of macrophages infected with B. pseudomallei by RNA-seq demonstrated upregulation of immune-associated genes, in particular a significant enrichment of genes in the TNF signaling pathway. Treatment of B. pseudomallei-infected macrophages with the Mip inhibitor, AN_CH_37 resulted in a 5.3-fold reduction of il1b when compared to cells treated with DMSO, which the inhibitors were solubilized in. A statistically significant reduction in IL-1β levels in culture supernatants was seen 24 hours post-infection with AN_CH_37, as well as other pro-inflammatory cytokines, namely IL-6 and TNF-α. Treatment with AN_CH_37 also reduced the survival of B. pseudomallei in macrophages after 24 hours which was accompanied by a significant reduction in B. pseudomallei-induced cytotoxicity as determined by lactate dehydrogenase release.DiscussionThese data highlight the potential to utilize Mip inhibitors in reducing potentially harmful pro-inflammatory responses resulting from B. pseudomallei infection in macrophages. This could be of significance since overstimulation of pro-inflammatory responses can result in immunopathology, tissue damage and septic shock.

Project description:Burkholderia pseudomallei is the causative agent of melioidosis, which is endemic primarily in Southeast Asia and northern Australia but is increasingly being seen in other tropical and subtropical regions of the world. Melioidosis is associated with high morbidity and mortality rates, which is mediated by the wide range of virulence factors encoded by B. pseudomallei. These virulence determinants include surface polysaccharides such as lipopolysaccharide (LPS) and capsular polysaccharides (CPS). Here, we investigated a predicted arabinose-5-phosphate isomerase (API) similar to KdsD in B. pseudomallei strain K96243. KdsD is required for the production of the highly conserved 3-deoxy-d-manno-octulosonic acid (Kdo), a key sugar in the core region of LPS. Recombinant KdsD was expressed and purified, and API activity was determined. Although a putative API paralogue (KpsF) is also predicted to be encoded, the deletion of kdsD resulted in growth defects, loss of motility, reduced survival in RAW 264.7 murine macrophages, and attenuation in a BALB/c mouse model of melioidosis. Suppressor mutations were observed during a phenotypic screen for motility, revealing single nucleotide polymorphisms or indels located in the poorly understood CPS type IV cluster. Crucially, suppressor mutations did not result in reversion of attenuation in vivo. This study demonstrates the importance of KdsD for B. pseudomallei virulence and highlights further the complex nature of the polysaccharides it produces. IMPORTANCE The intrinsic resistance of B. pseudomallei to many antibiotics complicates treatment. This opportunistic pathogen possesses a wide range of virulence factors, resulting in severe and potentially fatal disease. Virulence factors as targets for drug development offer an alternative approach to combat pathogenic bacteria. Prior to initiating early drug discovery approaches, it is important to demonstrate that disruption of the target gene will prevent the development of disease. This study highlights the fact that KdsD is crucial for virulence of B. pseudomallei in an animal model of infection and provides supportive phenotypic characterization that builds a foundation for future therapeutic development.

Dataset Information

A Burkholderia pseudomallei macrophage infectivity potentiator-like protein has rapamycin-inhibitable peptidylprolyl isomerase activity and pleiotropic effects on virulence.

Publications

A Burkholderia pseudomallei macrophage infectivity potentiator-like protein has rapamycin-inhibitable peptidylprolyl isomerase activity and pleiotropic effects on virulence.

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