Oleg Krut

Large scale multiplex PCR improves pathogen detection by DNA microarrays

BackgroundMedium density DNA microchips that carry a collection of probes for a broad spectrum of pathogens, have the potential to be powerful tools for simultaneous species identification, detection of virulence factors and antimicrobial resistance determinants. However, their widespread use in microbiological diagnostics is limited by the problem of low pathogen numbers in clinical specimens revealing relatively low amounts of pathogen DNA.ResultsTo increase the detection power of a fluorescence-based prototype-microarray designed to identify pathogenic microorganisms involved in sepsis, we propose a large scale multiplex PCR (LSplex PCR) for amplification of several dozens of gene-segments of 9 pathogenic species. This protocol employs a large set of primer pairs, potentially able to amplify 800 different gene segments that correspond to the capture probes spotted on the microarray. The LSplex protocol is shown to selectively amplify only the gene segments corresponding to the specific pathogen present in the analyte. Application of LSplex increases the microarray detection of target templates by a factor of 100 to 1000.ConclusionOur data provide a proof of principle for the improvement of detection of pathogen DNA by microarray hybridization by using LSplex PCR.

Loss of Dictyostelium ATG9 results in a pleiotropic phenotype affecting growth, development, phagocytosis and clearance and replication of Legionella pneumophila

Infection of Dictyostelium discoideum with Legionella pneumophila resulted in a large number of differentially regulated genes among them three core autophagy genes, ATG8, ATG9 and ATG16. Macroautophagy contributes to many physiological and pathological processes and might also constitute an important mechanism in cell‐autonomous immunity. For further studies we selected the highly conserved ATG9. In colocalization studies with GFP‐tagged ATG9 and different organelle marker proteins we neither observed colocalization with mitochondria, the ER nor lysosomes. However, there was partial colocalization with the Golgi apparatus and many ATG9‐GFP‐containing vesicles localized along microtubules and accumulated around the microtubule organizing centre. ATG9‐deficient cells had pleiotropic defects. In addition to growth defects they displayed severe developmental defects, consistent with the known role of autophagy in Dictyostelium development. Unexpectedly, the ATG9 mutant also had a strong phagocytosis defect that was particularly apparent when infecting the cells with L. pneumophila. However, those Legionellae that entered the host could multiply better in mutant than in wild‐type cells, because of a less efficient clearance in the early and a more efficient replication in the late phase of infection. We conclude that ATG9 and hence macroautophagy has a protective role during pathogen infection.

Recent Zika Virus Isolates Induce Premature Differentiation of Neural Progenitors in Human Brain Organoids

The recent Zika virus (ZIKV) epidemic is associated with microcephaly in newborns. Although the connection between ZIKV and neurodevelopmental defects is widely recognized, the underlying mechanisms are poorly understood. Here we show that two recently isolated strains of ZIKV, an American strain from an infected fetal brain (FB-GWUH-2016) and a closely-related Asian strain (H/PF/2013), productively infect human iPSC-derived brain organoids. Both of these strains readily target to and replicate in proliferating ventricular zone (VZ) apical progenitors. The main phenotypic effect was premature differentiation of neural progenitors associated with centrosome perturbation, even during early stages of infection, leading to progenitor depletion, disruption of the VZ, impaired neurogenesis, and cortical thinning. The infection pattern and cellular outcome differ from those seen with the extensively passaged ZIKV strain MR766. The structural changes we see after infection with these more recently isolated viral strains closely resemble those seen in ZIKV-associated microcephaly.

Identification of Staphylococcus aureus exotoxins by combined sodium dodecyl sulfate gel electrophoresis and matrix-assisted laser desorption/ ionization-time of flight mass spectrometry.

Staphylococcus aureus is an important human pathogen whose pathogenesis involves the synthesis of cell wall associated virulence factors and secreted toxins with damaging effects on the host cells. Most of these pathogenic factors are synthesized in a growth‐phase dependent manner as a response to environmental stress like heat, lack of nutrients or other deleterious conditions. Conventional identification of these pathogenic factors is based on Western blot analysis or enzyme‐linked immunosorbent assay (ELISA) and is limited by the commercial availability of antibodies against these toxins. We report here the use of matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometry for monitoring the pathogenic factors of S. aureus. For the identification of pathogenic factors, a methicillin sensitive strain of S. aureus, ATCC‐29213, was grown at 37°C or 42°C in brain‐heart infusion broth and harvested during the early stationary phase of growth. Secreted proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, enzymatically digested with trypsin and analyzed by MALDI‐TOF mass spectrometry. When grown at 42°C, alpha‐ and beta‐hemolysins were found to accumulate in S. aureus supernatants while the concentration of protein A was slightly decreased. The identity of some of these toxins was confirmed by Western‐blot analysis. MALDI‐TOF mass spectrometry combined with sodium dodecyl sulfate gel electrophoresis represents a rapid and simple approach to characterize the virulence of S. aureus strains which seems to be particularly valuable for the identification of S. aureus exotoxins for which ELISA is not established.

Expression of lysostaphin in HeLa cells protects from host cell killing by intracellular Staphylococcus aureus

The Staphylococcus aureus-specific cell wall endopeptidase lysostaphin was used as a model for an intracellular acting bactericidal antibiotic. HeLa cells were transfected with an expression vector directing the heterologous expression of lysostaphin in the cytoplasm. Expression, subcellular localization and enzymatic activity of lysostaphin were investigated by immunoblotting, fluorescent microscopy and agar diffusion assays. Both transiently and stably transfected HeLa cells showed a strong expression of active lysostaphin. After infection with S. aureus, the intracellular number of S. aureus and the host cell viability were determined. This staphylolytic activity resulted in a strong reduction of intracellular S. aureus in a time- and dose-dependent manner. Furthermore, host cells expressing lysostaphin became protected from S. aureus-induced cell death. Our data demonstrate the potential of intracellularly acting cell-wall active drugs or antibiotics that kill S. aureus without causing harm to the infected host cells.

Diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with tigecycline resistance in Acinetobacter baumannii

ObjectivesnTo investigate the mechanisms of tigecycline resistance in isogenic Acinetobacter baumannii isolate pairs as well as 65 unique clinical A. baumannii isolates obtained during the MagicBullet clinical trial from Greece, Italy and Spain.nnnMethodsnA. baumannii isolates were subjected to WGS and the regulatory genes of resistance-nodulation-cell division (RND)-type efflux pumps were analysed. MICs were determined by agar dilution and the expression of RND-type efflux pumps was measured by semi-quantitative RT-PCR.nnnResultsnIn isolate pairs, disruption of adeS or adeN by ISs increased adeB or adeJ expression and conferred increased resistance to at least three antimicrobial classes, respectively. The insertion of ISAba1 in adeN was observed in more than 30% of tested isolates and was the most prevalent IS. Furthermore, the insertion of ISAba125 and ISAba27 into adeN was observed for the first time in A. baumannii isolates. Besides ISs, several different mutations were observed in adeN (e.g. deletions and premature stop codons), all of which led to increased tigecycline MICs. Moreover, several amino acid substitutions were detected in AdeRS, AdeN and AdeL. Of note, the substitutions D21V, G25S and D26N in AdeR were found in multiple sequences and suggest a mutational hotspot.nnnConclusionsnThis study provides an insight into the different mechanisms associated with tigecycline resistance using a genomic approach and points out the importance of considering adeRS and adeN as markers for tigecycline-resistant A. baumannii isolates.