Ivo Steinmetz

Small-Colony Variants of Pseudomonas aeruginosa in Cystic Fibrosis

In the context of chronic lung infection due to Pseudomonas aeruginosa in cystic fibrosis (CF), attention has been focused on the presence of the most common mucoid phenotype. In this study, the presence of small-colony variants (SCVs) of P. aeruginosa in respiratory tract specimens from patients with CF was investigated, and the clinical conditions predisposing to SCVs were analyzed. P. aeruginosa SCVs were isolated from 33 of 86 P. aeruginosa-positive CF patients over a 2-year period. Fast-growing revertants with larger surface colonies could be isolated from SCV populations. Electron microscopy revealed no significant difference in cell size or morphology. MICs of a broad range of antipseudomonas agents for SCVs were two- to eightfold higher than values for revertants. Recovery of SCVs was correlated with parameters revealing poor lung function and was significantly associated with daily inhalation of tobramycin or colistin.

Identification of Burkholderia pseudomallei Genes Required for the Intracellular Life Cycle and In Vivo Virulence

ABSTRACT The bacterial pathogen Burkholderia pseudomallei invades host cells, escapes from endocytic vesicles, multiplies intracellularly, and induces the formation of actin tails and membrane protrusions, leading to direct cell-to-cell spreading. This study was aimed at the identification of B. pseudomallei genes responsible for the different steps of this intracellular life cycle. B. pseudomallei transposon mutants were screened for a reduced ability to form plaques on PtK2 cell monolayers as a result of reduced intercellular spreading. Nine plaque assay mutants with insertions in different open reading frames were selected for further studies. One mutant defective in a hypothetical protein encoded within the Bsa type III secretion system gene cluster was found to be unable to escape from endocytic vesicles after invasion but still multiplied within the vacuoles. Another mutant with a defect in a putative exported protein reached the cytoplasm but exhibited impaired actin tail formation in addition to a severe intracellular growth defect. In four mutants, the transposon had inserted into genes involved in either purine, histidine, or p-aminobenzoate biosynthesis, suggesting that these pathways are essential for intracellular growth. Three mutants with reduced plaque formation were shown to have gene defects in a putative cytidyltransferase, a putative lipoate-protein ligase B, and a hypothetical protein. All nine mutants proved to be significantly attenuated in a murine model of infection, with some mutants being essentially avirulent. In conclusion, we have identified a number of novel major B. pseudomallei virulence genes which are essential for the intracellular life cycle of this pathogen.

Killing of Caenorhabditis elegans by Burkholderia cepacia is controlled by the cep quorum-sensing system

Burkholderia cepacia H111, which was isolated from a cystic fibrosis patient, effectively kills the nematode Caenorhabditis elegans. Depending on the medium used for growth of the bacterium two different killing modes were observed. On high‐osmolarity medium the nematodes became paralysed and died within 24u2003h. Using filter assays we provide evidence that this killing mode involves the production of an extracellular toxin. On nematode growth medium killing occurs over the course of 2–3u2003days and involves the accumulation of bacteria in the intestinal lumen of C. elegans. We demonstrate that the cep quorum‐sensing system of H111 is required for efficient killing of C. elegans under both killing conditions. Using the C. elegans phm‐2 mutant that has a non‐functional grinder evidence is provided that the cep system is required to enter the intestinal lumen but is dispensable for the colonization of the gut. Furthermore, we demonstrate that the type II secretion machinery is not essential for nematode killing.

Role of Inducible Nitric Oxide Synthase and NADPH Oxidase in Early Control of Burkholderia pseudomallei Infection in Mice

ABSTRACT Infection with the soil bacterium Burkholderia pseudomallei can result in a variety of clinical outcomes, including asymptomatic infection. The initial immune defense mechanisms which might contribute to the various outcomes after environmental contact with B. pseudomallei are largely unknown. We have previously shown that relatively resistant C57BL/6 mice can restrict bacterial B. pseudomallei growth more efficiently within 1 day after infection than highly susceptible BALB/c mice. By using this model, our study aimed to investigate the role of macrophage-mediated effector mechanisms during early B. pseudomallei infection. Depletion of macrophages revealed an essential role of these cells in the early control of infection in BALB/c and C57BL/6 mice. Strikingly, the comparison of the anti-B. pseudomallei activity of bone marrow-derived macrophages (BMM) from C57BL/6 and BALB/c mice revealed an enhanced bactericidal activity of C57BL/6 BMM, particularly after gamma interferon (IFN-γ) stimulation. In vitro experiments with C57BL/6 gp91phox−/− BMM showed an impaired intracellular killing of B. pseudomallei compared to experiments with wild-type cells, although C57BL/6 gp91phox−/− cells still exhibited substantial killing activity. The anti-B. pseudomallei activity of C57BL/6 iNOS−/− BMM was not impaired. C57BL/6 gp91phox−/− mice lacking a functional NADPH oxidase were more susceptible to infection, whereas C57BL/6 mice lacking inducible nitric oxide synthase (iNOS) did not show increased susceptibility but were slightly more resistant during the early phase of infection. Thus, our data suggest that IFN-γ-mediated but iNOS-independent anti-B. pseudomallei mechanisms of macrophages might contribute to the enhanced resistance of C57BL/6 mice compared to that of BALB/c mice in the early phase of infection.

Expression Analysis of a Highly Adherent and Cytotoxic Small Colony Variant of Pseudomonas aeruginosa Isolated from a Lung of a Patient with Cystic Fibrosis

The heterogeneous environment of the lung of the cystic fibrosis (CF) patient gives rise to Pseudomonas aeruginosa small colony variants (SCVs) with increased antibiotic resistance, autoaggregative growth behavior, and an enhanced ability to form biofilms. In this study, oligonucleotide DNA microarrays were used to perform a genome-wide expression study of autoaggregative and highly adherent P. aeruginosa SCV 20265 isolated from a CF patients lung in comparison with its clonal wild type and a revertant generated in vitro from the SCV population. Most strikingly, SCV 20265 showed a pronounced upregulation of the type III protein secretion system (TTSS) and the respective effector proteins. This differential expression was shown to be biologically meaningful, as SCV 20265 and other hyperpiliated and autoaggregative SCVs with increased TTSS expression were significantly more cytotoxic for macrophages in vitro and were more virulent in a mouse model of respiratory tract infection than the wild type. The observed cytotoxicity and virulence of SCV 20265 required exsA, an important transcriptional activator of the TTSS. Thus, the prevailing assumption that P. aeruginosa is subject to selection towards reduced cytotoxicity and attenuated virulence during chronic CF lung infection might not apply to all clonal variants.

Inter- and Intraclonal Diversity of the Pseudomonas aeruginosa Proteome Manifests within the Secretome

The proteomes of cultured Pseudomonas aeruginosa isolates from chronically infected cystic fibrosis (CF) lungs were compared by using genetically divergent clones and isogenic morphotypes of one strain. Cellular extracts gave very similar protein patterns in two-dimensional gels, suggesting that the conserved species-specific core genome encodes proteins that are expressed under standard culture conditions in vitro. In contrast, the protein profiles of extracts of culture supernatants were dependent on the growth phase, and there were significant differences between clones. The profiles also varied within clonally related morphotypes from one CF patient, including a hyperpiliated small-colony variant. Mass spectrometry revealed that this variant overexpressed proteins secreted by the type I secretion system (including proteins involved in iron acquisition) and by the type III secretion system. Furthermore, the proteins in the supernatant extracts from the small-colony variant which were recognized by sera from different CF patients varied greatly. We concluded that the secretome expression is a sensitive measure of P. aeruginosa strain variation.

Combination of rRNA-Targeted Hybridization Probes and Immuno-Probes for the Identification of Bacteria by Flow Cytometry

Summary Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) were for the first time combined for the flow cytometric identification of bacteria. Artificial mixtures of fixed cells were hybridized with fluorescein-labeled, rRNA-targeted oligonucleotide probes and stained indirectly with biotinylated antibodies and R-phycoerythrin (PE) conjugated streptavidin. Finally, they were counterstained with 4,6diamidino-2-phenylindole (DAPI) in order to discriminate cells from background. Forward scatter, fluorescein- as well as PE- and DAPI-fluorescence were measured simultaneously for the differentiation of bacterial species by multiparameter flow cytometric analysis. The influence of detergents in the hybridization buffer on nonspecific antibody binding was evaluated. Sodium dodecyl sulfate (SDS) induced strong nonspecific staining and was, therefore, replaced by Tween 20. We found that the immunostaining steps can be performed before or after hybridization. This combination of rRNA-targeted hybridization probes and immuno-probes for flow cytometry makes possible the highly specific and automated identification of micro-organisms at any desired taxonomic level.

Detrimental Role of CC Chemokine Receptor 4 in Murine Polymicrobial Sepsis

ABSTRACT CC chemokine receptor 4 (CCR4) and its two ligands, CCL17 and CCL22, are critically involved in different immune processes. In models of lipopolysaccharide-induced shock, CCR4-deficient (CCR4−/−) mice showed improved survival rates associated with attenuated proinflammatory cytokine release. Using CCR4−/− mice with a C57BL/6 background, this study describes for the first time the role of CCR4 in a murine model of polymicrobial abdominal sepsis, the colon ascendens stent peritonitis (CASP). CASP-induced sepsis led to a massive downregulation of CCR4 in lymphoid and nonlymphoid tissues, whereas the expression of CCL17 and CCL22 was independent of the presence of CCR4. After CASP, CCR4−/− animals showed a strongly enhanced bacterial clearance in several organs but not in the peritoneal lavage fluid and the blood. In addition, significantly reduced levels of proinflammatory cytokines/chemokines were measured in organ supernatants as well as in the sera of CCR4−/− mice. CCR4 deficiency consequently resulted in an attenuated severity of systemic sepsis and a strongly improved survival rate after CASP or CASP with intervention. Thus, our data provide clear evidence that CCR4 plays a strictly detrimental role in the course of polymicrobial sepsis.

Defense Mechanisms of Hepatocytes Against Burkholderia pseudomallei

The Gram-negative facultative intracellular rod Burkholderia pseudomallei causes melioidosis, an infectious disease with a wide range of clinical presentations. Among the observed visceral abscesses, the liver is commonly affected. However, neither this organotropism of B. pseudomallei nor local hepatic defense mechanisms have been thoroughly investigated so far. Own previous studies using electron microscopy of the murine liver after systemic infection of mice indicated that hepatocytes might be capable of killing B. pseudomallei. Therefore, the aim of this study was to further elucidate the interaction of B. pseudomallei with these cells and to analyze the role of hepatocytes in anti-B. pseudomallei host defense. In vitro studies using the human hepatocyte cell line HepG2 revealed that B. pseudomallei can invade these cells. Subsequently, B. pseudomallei is able to escape from the vacuole, to replicate within the cytosol of HepG2 cells involving its type 3 and type 6 secretion systems, and to induce actin tail formation. Furthermore, stimulation of HepG2 cells showed that IFNγ can restrict growth of B. pseudomallei in the early and late phase of infection whereas the combination of IFNγ, IL-1β, and TNFα is required for the maximal antibacterial activity. This anti-B. pseudomallei defense of HepG2 cells did not seem to be mediated by inducible nitric oxide synthase-derived nitric oxide or NADPH oxidase-derived superoxide. In summary, this is the first study describing B. pseudomallei intracellular life cycle characteristics in hepatocytes and showing that IFNγ-mediated, but nitric oxide- and reactive oxygen species-independent, effector mechanisms are important in anti-B. pseudomallei host defense of hepatocytes.

Evidence for Polyadenylated mRNA in Pseudomonas aeruginosa

In this paper, we report the synthesis of Pseudomonas aeruginosa cDNA in the presence of oligo(dT) primers. Hybridization of oligonucleotide DNA microarrays indicates that under the experimental conditions used, at least 43.7% of the expressed genes from P. aeruginosa PAO1, representing many different functional classes, can be detected by using oligo(dT)-primed cDNAs.