mRNA-based approach to monitor recombinant gamma-interferon restoration of LPS-induced endotoxin tolerance.

INTRODUCTION: It is now well accepted that sepsis is associated with the development of a pronounced immunosuppressive state, characterized by severe immune alterations (e.g. reduced proliferative capacity, endotoxin tolerance, apoptosis) participating in increased mortality and susceptibility to nosocomial infections. Efforts are currently aimed at restoring a functional immune response in septic patients. Successful therapy depends on the identification of appropriate immunostimulatory drugs and on the development of suitable biomarkers that could be used to stratify patients and to follow response to treatment. METHODS: In this study, we evaluated the ex vivo effect of recombinant interferon gamma (rIFN-γ) in restoring monocyte functionality (endotoxin-induced Tumor Necrosis Factor-α production) in a two-hit model of endotoxin tolerance (ET) with peripheral blood mononuclear cells from healthy volunteers and in whole blood of septic shock patients. Importantly, we used quantitative-reverse transcription polymerase-chain reaction to monitor the effect of rIFN-γ on the expression of seven genes known to participate in ET (TNF-α, IL-10, HLA-DRA, CIITA, IRAK-M, ABIN-3 and LY64). RESULTS: Expression analysis of those genes confirmed the presence of an immunosuppression state and the ex vivo restoration of immune functions by rIFN-γ. We show for the first time that rIFN-γ is able to bypass, at the mRNA level, the effect of negative regulators of the LPS signalling pathway such as IRAK-M, ABIN-3 and LY64. CONCLUSIONS: Overall, mRNA expressions of a panel of genes could represent promising candidates for the ex vivo evaluation of rIFN-γ effect on monocyte functionality. This ex vivo translational research study demonstrates the potential of a mRNA-based approach to successfully monitor drug efficacy.

HU protein affects transcription of surface polysaccharide synthesis genes in Porphyromonas gingivalis.

K-antigen capsule synthesis is an important virulence determinant of the oral anaerobe Porphyromonas gingivalis. We previously reported that the locus required for synthesis of this surface polysaccharide in strain W83 (TIGR identification PG0106 to PG0120) is transcribed as a large (∼16.7-kb) polycistronic message. Through sequence analysis, we have now identified a 77-bp inverted repeat located upstream (206 bp) of the start codon of PG0106 that is capable of forming a large hairpin structure. Further sequence analysis just upstream and downstream of the capsule synthesis genes revealed the presence of two genes oriented in the same direction as the operon that are predicted to encode DNA binding proteins: PG0104, which is highly similar (57%) to DNA topoisomerase III, and PG0121, which has high similarity (72%) to DNA binding protein HU (ß-subunit). In this report, we show that these two genes, as well as the 77-bp inverted repeat region, are cotranscribed with the capsule synthesis genes, resulting in a large transcript that is ∼19.4 kb (based on annotation). We also show that a PG0121 recombinant protein is a nonspecific DNA binding protein with strong affinity to the hairpin structure, in vitro, and that transcript levels of the capsule synthesis genes are downregulated in a PG0121 deletion mutant. Furthermore, we show that this decrease in transcript levels corresponds to a decrease in the amount of polysaccharide produced. Interestingly, expression analysis of another polysaccharide synthesis locus (PG1136 to PG1143) encoding genes involved in synthesis of a surface-associated phosphorylated branched mannan (APS) indicated that this locus is also downregulated in the PG0121 mutant. Altogether our data indicate that HU protein modulates expression of surface polysaccharides in P. gingivalis strain W83.

Differential function of Listeria monocytogenes listeriolysin O and phospholipases C in vacuolar dissolution following cell-to-cell spread.

We investigated the role of listeriolysin O (LLO) and the bacterial phospholipases PI-PLC and PC-PLC in cell-to-cell spread of Listeria monocytogenes. We showed that LLO is essential for cell-to-cell spread in primary murine macrophages. Electron micrographs revealed that in the absence of continued LLO expression, bacteria remain trapped in secondary spreading vacuoles having either a double or single membrane. In bacteria lacking PI-PLC and PC-PLC, cessation of LLO expression after initiation of infection resulted in a significant increase in the proportion of bacteria trapped in double-membrane compartments. We propose that the bacterial phospholipases are involved in the dissolution of the inner membrane of the spreading vacuole, yet are not sufficient for disruption of the outer membrane. As a consequence, we identified LLO as a key factor in the disruption of the outer membrane. This model is consistent with the observation that LLO is dispensable for cell-to-cell spread from human macrophages into a cell type in which LLO is not required for vacuolar escape. These data suggest that during human infection, spreading of L. monocytogenes to distant organs is likely to occur even in the absence of LLO expression, and that the bacterial phospholipases may be sufficient to mediate continued cell-to-cell spread.

Directed antigen delivery as a vaccine strategy for an intracellular bacterial pathogen.

We have developed a vaccine strategy for generating an attenuated strain of an intracellular bacterial pathogen that, after uptake by professional antigen-presenting cells, does not replicate intracellularly and is readily killed. However, after degradation of the vaccine strain within the phagolysosome, target antigens are released into the cytosol for endogenous processing and presentation for stimulation of CD8(+) effector T cells. Applying this strategy to the model intracellular pathogen Listeria monocytogenes, we show that an intracellular replication-deficient vaccine strain is cleared rapidly in normal and immunocompromised animals, yet antigen-specific CD8(+) effector T cells are stimulated after immunization. Furthermore, animals immunized with the intracellular replication-deficient vaccine strain are resistant to lethal challenge with a virulent WT strain of L. monocytogenes. These studies suggest a general strategy for developing safe and effective, attenuated intracellular replication-deficient vaccine strains for stimulation of protective immune responses against intracellular bacterial pathogens.

Cytolysin-dependent delay of vacuole maturation in macrophages infected with Listeria monocytogenes.

The bacterial pathogen Listeria monocytogenes (Lm) evades the antimicrobial mechanisms of macrophages by escaping from vacuoles to the cytosol, through the action of the cytolysin listeriolysin O (LLO). Because of heterogeneities in the timing and efficiency of escape, important questions about the contributions of LLO to Lm vacuole identity and trafficking have been inaccessible. Expression of cyan fluorescent protein (CFP)-labelled endocytic membrane markers in macrophages along with a yellow fluorescent protein (YFP)-labelled indicator of Lm entry to the cytosol identified compartments lysed by bacteria. Lm escaped from Rab5a-negative, lysosome-associated membrane protein-1 (LAMP1)-negative, Rab7-positive, phosphatidylinositol 3-phosphate [PI(3)P]-positive vacuoles. Lm vacuoles did not label with YFP-Rab5a unless the bacteria were first opsonized with IgG. Wild-type Lm delayed vacuole fusion with LAMP1-positive lysosomes, relative to LLO-deficient Lm. Bacteria prevented from expressing LLO until their arrival into LAMP1-positive lysosomes escaped inefficiently. Thus, the LLO-dependent delay of Lm vacuole fusion with lysosomes affords Lm a competitive edge against macrophage defences by providing bacteria more time in organelles they can penetrate.

Identification of potential cell-surface proteins in Candida albicans and investigation of the role of a putative cell-surface glycosidase in adhesion and virulence.

Cell-surface proteins are attractive targets for the development of novel antifungals as they are more accessible to drugs than are intracellular targets. By using a computational biology approach, we identified 180 potential cell-surface proteins in Candida albicans, including the known cell-surface adhesin Als1 and other cell-surface antigens, such as Pra1 and Csa1. Six proteins (named Csf1-6 for cell-surface factors) were selected for further biological characterization. First, we verified that the selected CSF genes are expressed in the yeast and/or hyphal form and then we investigated the effect of the loss of each CSF gene on cell-wall integrity, filamentation, adhesion to mammalian cells and virulence. As a result, we identified Csf4, a putative glycosidase with an apparent orthologue in Saccharomyces cerevisiae (Utr2), as an important factor for cell-wall integrity and maintenance. Interestingly, deletion of CSF4 also resulted in a defect in filamentation, a reduction in adherence to mammalian cells in an in vitro adhesion assay, and a prolongation of survival in an immunocompetent mouse model of disseminated candidiasis. A delay in colonization of key organs (e.g. kidney) was also observed, which is consistent with a reduction in virulence of the csf4-deletion strain. These data indicate a key role for extracellular glycosidases in fungal pathogenesis and represent a new site for therapeutic intervention to cure and prevent fungal disease.