Yersinia enterocolitica employs a type three secretion system (T3SS) to translocate immunosuppressive effector proteins into host cells. To this end, the T3SS assembles a translocon/pore complex ...composed of the translocator proteins YopB and YopD in host cell membranes serving as an entry port for the effectors. The translocon is formed in a Yersinia-containing pre-phagosomal compartment that is connected to the extracellular space. As the phagosome matures, the translocon and the membrane damage it causes are recognized by the cell-autonomous immune system. We infected cells in the presence of fluorophore-labeled ALFA-tag-binding nanobodies with a Y. enterocolitica strain expressing YopD labeled with an ALFA-tag. Thereby we could record the integration of YopD into translocons and its intracellular fate in living host cells. YopD was integrated into translocons around 2 min after uptake of the bacteria into a phosphatidylinositol-4,5-bisphosphate enriched pre-phagosomal compartment and remained there for 27 min on average. Damaging of the phagosomal membrane as visualized with recruitment of GFP-tagged galectin-3 occurred in the mean around 14 min after translocon formation. Shortly after recruitment of galectin-3, guanylate-binding protein 1 (GBP-1) was recruited to phagosomes, which was accompanied by a decrease in the signal intensity of translocons, suggesting their degradation or disassembly. In sum, we were able for the first time to film the spatiotemporal dynamics of Yersinia T3SS translocon formation and degradation and its sensing by components of the cell-autonomous immune system.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Early and adequate antimicrobial therapy has been shown to improve the clinical outcome in bloodstream infections (BSI). To provide rapid pathogen identification for targeted treatment, we applied ...matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry fingerprinting to bacteria directly recovered from blood culture bottles. A total of 304 aerobic and anaerobic blood cultures, reported positive by a Bactec 9240 system, were subjected in parallel to differential centrifugation with subsequent mass spectrometry fingerprinting and reference identification using established microbiological methods. A representative spectrum of bloodstream pathogens was recovered from 277 samples that grew a single bacterial isolate. Species identification by direct mass spectrometry fingerprinting matched reference identification in 95% of these samples and worked equally well for aerobic and anaerobic culture bottles. Application of commonly used score cutoffs to classify the fingerprinting results led to an identification rate of 87%. Mismatching mostly resulted from insufficient bacterial numbers and preferentially occurred with Gram-positive samples. The respective spectra showed low concordance to database references and were effectively rejected by score thresholds. Spiking experiments and examination of the respective study samples even suggested applicability of the method to mixed cultures. With turnaround times around 100 min, the approach allowed for reliable pathogen identification at the day of blood culture positivity, providing treatment-relevant information within the critical phase of septic illness.
In 2011 northern Germany experienced a large outbreak of Shiga-Toxigenic Escherichia coli O104:H4. The large amount of samples sent to microbiology laboratories for epidemiological assessment ...highlighted the importance of fast and inexpensive typing procedures. We have therefore evaluated the applicability of a MALDI-TOF mass spectrometry based strategy for outbreak strain identification.
Specific peaks in the outbreak strain's spectrum were identified by comparative analysis of archived pre-outbreak spectra that had been acquired for routine species-level identification. Proteins underlying these discriminatory peaks were identified by liquid chromatography tandem mass spectrometry and validated against publicly available databases. The resulting typing scheme was evaluated against PCR genotyping with 294 E. coli isolates from clinical samples collected during the outbreak.
Comparative spectrum analysis revealed two characteristic peaks at m/z 6711 and m/z 10883. The underlying proteins were found to be of low prevalence among genome sequenced E. coli strains. Marker peak detection correctly classified 292 of 293 study isolates, including all 104 outbreak isolates.
MALDI-TOF mass spectrometry allowed for reliable outbreak strain identification during a large outbreak of Shiga-Toxigenic E. coli. The applied typing strategy could probably be adapted to other typing tasks and might facilitate epidemiological surveys as part of the routine pathogen identification workflow.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract Early detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) and initiation of adequate infection control measures are important objectives in hospital hygiene. To ...reach these goals, prompt determination of epidemiologic relatedness of clinical MRSA isolates is essential. Genetic typing methods like pulsed-field gel electrophoresis (PFGE), spa typing, or multilocus sequence typing (MLST) have a high discriminatory power, however, these methods are time consuming and cost intensive. The aim of this study was to investigate the potential of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for discrimination of major MRSA lineages. By analysis of mass spectra from 25 representative MRSA isolates belonging to the 5 major hospital-acquired (HA) MRSA clonal complexes (CC5, CC8, CC22, CC30, CC45; deduced from spa typing), reproducible spectrum differences were observed at 13 characteristic m / z values allowing robust discrimination of the clonal complexes. When 60 independent clinical MRSA isolates were tested for the presence or absence of the 13 characteristic MALDI-TOF MS peaks, 15 different profiles (MALDI types) could be detected. Hierarchical clustering of the MALDI types showed high concordance with the clonal complexes. Our results suggest that MALDI-TOF MS has the potential to become a valuable first-line tool for inexpensive and rapid typing of MRSA in infection control.
Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery-also named injectisome-assembles a pore ...complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their formation have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualize Y. enterocolitica translocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology, we provide novel information about the spatiotemporal organization of T3SS translocons and their regulation by host cell factors.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Globally,
is an important bacterial pathogen causing a wide range of community and hospital acquired infections. In Ghana, resistance of
to locally available antibiotics is increasing but the ...molecular basis of resistance and the population structure of
in particular in chronic wounds are poorly described. However, this information is essential to understand the underlying mechanisms of resistance and spread of resistant clones. We therefore subjected 28
isolates from chronic infected wounds in a rural area of Ghana to whole genome sequencing.
Overall, resistance of
to locally available antibiotics was high and 29% were Methicillin resistant
(MRSA). The most abundant sequence type was ST88 (29%, 8/28) followed by ST152 (18%, 5/28). All ST88 carried the
gene, which was associated with this sequence type only. Chloramphenicol resistance gene
was exclusively associated with the methicillin-resistant ST88 strains. Panton-Valentine leukocidin (PVL) carriage was associated with ST121 and ST152. Other detected mechanisms of resistance included
, conferring resistance to trimethoprim.
This study provides valuable information for understanding the population structure and resistance mechanisms of
isolated from chronic wound infections in rural Ghana.
Super‐resolution fluorescence microscopy technologies developed over the past two decades have pushed the resolution limit for fluorescently labeled molecules into the nanometer range. These ...technologies have the potential to study bacterial structures, for example, macromolecular assemblies such as secretion systems, with single‐molecule resolution on a millisecond time scale. Here we review recent applications of super‐resolution fluorescence microscopy with a focus on bacterial secretion systems. We also describe MINFLUX fluorescence nanoscopy, a relatively new technique that promises to one day produce molecular movies of molecular machines in action.
Current developments in super‐resolution fluorescence microscopy are opening up new possibilities in molecular and cellular bacteriology. The latest applications are presented including the use of MINFLUX nanoscopy for research into secretion systems.
Purpose
Hypertoxigenic
Streptococcus pyogenes emm
1 lineage M1
UK
has recently been associated with upsurges of invasive infections and scarlet fever in several countries, but whole-genome sequencing ...surveillance data of lineages circulating in Germany is lacking. In this study, we investigated recent iGAS isolates from our laboratory at a German tertiary care center for the presence of the M1
UK
lineage.
Methods
Whole-genome sequencing was employed to characterize a collection of 47 consecutive non-copy isolates recovered from blood cultures (21) and tissue samples (26) in our laboratory between October 2022 and April 2023.
Results
M protein gene (
emm
) typing distinguished 14 different
emm
types, with
emm
1 (17) being the dominant type. Single-nucleotide polymorphism (SNP) analysis confirmed the presence of all 27 SNPs characteristic for the M1
UK
lineage in 14 of 17
emm
1 isolates.
Conclusion
This study has shown for the first time that M1
UK
is present in Germany and might constitute a driving force in the observed surge of GAS infections. This observation mirrors developments in the UK and other countries and underscores the importance of WGS surveillance to understand the epidemiology of GAS.
Pathogenic bacteria of the genus Yersinia include Y. pestis-the agent of plaque-and two enteropathogens, Y. enterocolitica, and Y. pseudotuberculosis. These pathogens have developed an array of ...virulence factors aimed at manipulating Rho GTP-binding proteins and the actin cytoskeleton in host cells to cross the intestinal barrier and suppress the immune system. Yersinia virulence factors include outer membrane proteins triggering cell invasion by binding to integrins, effector proteins injected into host cells to manipulate Rho protein functions and a Rho protein-activating exotoxin. Here, we present an overview of how Yersinia and host factors are integrated in a regulatory network that orchestrates the subversion of host defense.
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