Live imaging of Yersinia translocon formation and immune recognition in host cells

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. 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.

Potential agrifood application of seriguela (Spondias purpurea L.) residues extract and nanoZnO as antimicrobial, antipathogenic and antivirulence agents

The impact of phytopathogenic microorganisms in several crops causes significant losses in agrifood industry, spoilage throughout food chain and storage. Nanoparticles and plant extracts have been highlighted by their antimicrobial properties applied in food packaging, agriculture, drug delivery systems and other medical approaches. Over the past few years, this group have studied the application of ZnO nanoparticles and agroindustrial wastes in edible food coatings/films. This study aimed to evaluate active characteristics from the extract of seriguela processing wastes and nanoZnO regarding to their inhibitory activity against bacterial pathogenicity and virulence systems TTSS (Type Three Secretion System) and QS (Quorum Sensing) for Pseudomonas savastanoi. Also, antibacterial action (inhibition area) against species of Curtobacterium, Clavibacter, E. coli, Xanthomonas and Serratia, and antifungal against Botrytis cinerea (reduction in colony size). The 60% extract inhibited the activation of QS and TTSS system in 20.26% and 13.54%, respectively; while nanoZnO at 3% reduced 46.77% QS and increased 302.88% TTSS. Extract without dilution inhibited the growth of Clavibacter michiganensis pv michiganensis (Gram-positive) and Xanthomonas phaseoli (Gram-negative), inhibitory zone of 94.25 mm2 and 452.39 mm2 respectively. The latter also being inhibited by nanoZnO 1 and 2% (138.23 mm2) and 3% (275.67 mm2). Pure extract inhibited 17.38% growth of fungal colony and nanoZnO (1 and 3%) in 33.08%. Finally, the active agents studied showed to be promising in the prevention of phytopathogenic diseases and consequently economic losses, food films/coatings and the extract as a biopesticide, reducing the environmental impact.

Comparative genomics of Chinese and international isolates of Escherichia albertii: population structure and evolution of virulence and antimicrobial resistance

Escherichia albertii is a recently recognized species in the genus Escherichia that causes diarrhoea. The population structure, genetic diversity and genomic features have not been fully examined. Here, 169 E. albertii isolates from different sources and regions in China were sequenced and combined with 312 publicly available genomes (from additional 14 countries) for genomic analyses. The E. albertii population was divided into two clades and eight lineages, with lineage 3 (L3), L5 and L8 more common in China. Clinical isolates were observed in all clades/lineages. Virulence genes were found to be distributed differently among lineages: subtypes of the intimin encoding gene eae and the cytolethal distending toxin gene cdtB were lineage associated, and the second type three secretion system (ETT2) island was truncated in L3 and L6. Seven new eae subtypes and one new cdtB subtype (cdtB-VI) were identified. Alarmingly, 85.9 % of the Chinese E. albertii isolates were predicted to be multidrug-resistant (MDR) with 35.9 % harbouring genes capable of conferring resistance to 10 to 14 different drug classes. The majority of the MDR isolates were of poultry source from China and belonged to four sequence types (STs) [ST4638, ST4479, ST4633 and ST4488]. Thirty-four plasmids with some carrying MDR and virulence genes, and 130 prophages were identified from 17 complete E. albertii genomes. The 130 intact prophages were clustered into five groups, with group five prophages harbouring more virulence genes. We further identified three E. albertii specific genes as markers for the identification of this species. Our findings provided fundamental insights into the population structure, virulence variation and drug resistance of E. albertii .

Delivering the pain: an overview of the type III secretion system with special consideration for aquatic pathogens

Gram-negative bacteria are known to subvert eukaryotic cell physiological mechanisms using a wide array of virulence factors, among which the type three-secretion system (T3SS) is often one of the most important. The T3SS constitutes a needle-like apparatus that the bacterium uses to inject a diverse set of effector proteins directly into the cytoplasm of the host cells where they can hamper the host cellular machinery for a variety of purposes. While the structure of the T3SS is somewhat conserved and well described, effector proteins are much more diverse and specific for each pathogen. The T3SS can remodel the cytoskeleton integrity to promote intracellular invasion, as well as silence specific eukaryotic cell signals, notably to hinder or elude the immune response and cause apoptosis. This is also the case in aquatic bacterial pathogens where the T3SS can often play a central role in the establishment of disease, although it remains understudied in several species of important fish pathogens, notably in Yersinia ruckeri. In the present review, we summarise what is known of the T3SS, with a special focus on aquatic pathogens and suggest some possible avenues for research including the potential to target the T3SS for the development of new anti-virulence drugs.

Identification of Novel Type Three Secretion System (T3SS) Inhibitors by Computational Methods and Anti-Salmonella Evaluations

Three type III secretion system (T3SS) inhibitors (compounds 5, 19, and 32) were identified by virtual screening and biological evaluation. These three compounds were evaluated against a panel of Salmonella species strains including S. enteritidis, S. typhi, S. typhimurium, S. paratyphi, and S. abortus equi, and their minimum inhibitory concentrations ranged from 1 to 53 μg/ml. Especially, these compounds showed comparable activity as the of the positive control gatifloxacin towards S. abortus equi. The present results suggest that these new T3SS inhibitors could be used as a potential lead molecule for drug development of anti-Salmonella.

The invasive pathogen Yersinia pestis disrupts host blood vasculature to spread and provoke hemorrhages

Yersinia pestis is a powerful pathogen with a rare invasive capacity. After a flea bite, the plague bacillus can reach the bloodstream in a matter of days giving way to invade the whole organism reaching all organs and provoking disseminated hemorrhages. However, the mechanisms used by this bacterium to cross and disrupt the endothelial vascular barrier remain poorly understood. In this study, an innovative model of in vivo infection was used to focus on the interaction between Y. pestis and its host vascular system. In the draining lymph nodes and in secondary organs, bacteria provoked the porosity and disruption of blood vessels. An in vitro model of endothelial barrier showed a role in this phenotype for the pYV/pCD1 plasmid that carries a Type Three Secretion System. This work supports that the pYV/pCD1 plasmid is responsible for the powerful tissue invasiveness capacity of the plague bacillus and the hemorrhagic features of plague.

Yersinia pseudotuberculosis YopE prevents uptake by M cells and instigates M cell extrusion in human ileal enteroid-derived monolayers.

Many pathogens use M cells to access the underlying Peyers patches and spread to systemic sites via the lymph as demonstrated by ligated loop murine intestinal models. However, the study of interactions between M cells and microbial pathogens has stalled due to the lack of cell culture systems. To overcome this obstacle, we use human ileal enteroid-derived monolayers containing five intestinal cell types including M cells to study the interactions between the enteric pathogen, Yersinia pseudotuberculosis (Yptb) and M cells. The Yptb type three secretion system (T3SS) effector Yops inhibit host defenses including phagocytosis and are critical for colonization of the intestine and Peyers patches. Therefore, it is not understood how Yptb traverses through M cells to breach the epithelium. By growing Yptb under two physiological conditions that mimic the early infectious stage (low T3SS-expression) or host-adapted stage (high T3SS-expression), we found that large numbers of Yptb specifically associated with M cells, recapitulating murine studies. Transcytosis through M cells was significantly higher by Yptb expressing low levels of T3SS, because YopE and YopH prevented Yptb uptake. YopE also caused M cells to extrude from the epithelium without inducing cell-death or disrupting monolayer integrity. Sequential infection with early infectious stage Yptb reduced host-adapted Yptb association with M cells. These data underscore the strength of enteroids as a model by discovering that Yops impede M cell function, indicating that early infectious stage Yptb more effectively penetrates M cells while the host may defend against M cell penetration of host-adapted Yptb.

The expression of virulence increases outer-membrane permeability and sensitivity to envelope stress in Salmonella Typhimurium

Environmental cues modulate the expression of virulence in bacterial pathogens. However, while cues that upregulate virulence are often intuitive and mechanistically well understood, this is less so for cues that downregulate virulence. In this study, we noticed that upregulation of the HilD virulence regulon in Salmonella Typhimurium (S.Tm) sensitized cells to membrane stress mediated by cholate, Tris/EDTA or heat. Further monitoring of membrane status and stress resistance of S.Tm cells in relation to virulence expression, revealed that co-expressed virulence factors embedded in the envelope (including the Type Three Secretion System 1 and the flagella) increased permeability, and stress sensitivity of the membrane. Importantly, pretreating the bacteria by sublethal stress inhibited virulence expression and restored stress resistance. As such, these results demonstrate a trade-off between virulence and stress resistance, which explains the downregulation of virulence expression in response to harsh environments in S.Tm.

Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

Type three secretion systems enable bacterial pathogens to inject effectors into the cytosol of eukaryotic hosts to reprogram cellular functions. It is technically challenging to label effectors and the secretion machinery without disrupting their structure/function. Herein, we present a new approach for labeling and visualization of previously intractable targets. Using genetic code expansion, we site-specifically labeled SsaP, the substrate specificity switch, and SifA, a here-to-fore unlabeled secreted effector. SsaP was secreted at later infection times; SsaP labeling demonstrated the stochasticity of injectisome and effector expression. SifA was labeled after secretion into host cells via fluorescent unnatural amino acids or non-fluorescent labels and a subsequent click reaction. We demonstrate the superiority of imaging after genetic code expansion compared to small molecule tags. It provides an alternative for labeling proteins that do not tolerate N- or C-terminal tags or fluorophores and thus is widely applicable to other secreted effectors and small proteins.