Direct Salmonella injection into enteroid cells allows the study of host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen-host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Salt- and Osmo-Responsive Sensor Histidine Kinases Activate the Bradyrhizobium diazoefficiens General Stress Response to Initiate Functional Symbiosis.

The general stress response (GSR) enables bacteria to sense and overcome a variety of environmental stresses. In alphaproteobacteria, stress-perceiving histidine kinases of the HWE and HisKA_2 families trigger a signaling cascade that leads to phosphorylation of the response regulator PhyR and, consequently, to activation of the GSR σ factor σEcfG. In the nitrogen-fixing bacterium Bradyrhizobium diazoefficiens, PhyR and σEcfG are crucial for tolerance against a variety of stresses under free-living conditions and also for efficient infection of its symbiotic host soybean. However, the molecular players involved in stress perception and activation of the GSR remained largely unknown. In this work, we first showed that a mutant variant of PhyR where the conserved phosphorylatable aspartate residue D194 was replaced by alanine (PhyRD194A) failed to complement the ΔphyR mutant in symbiosis, confirming that PhyR acts as a response regulator. To identify the PhyR-activating kinases in the nitrogen-fixing symbiont, we constructed in-frame deletion mutants lacking single, distinct combinations, or all of the 11 predicted HWE and HisKA_2 kinases, which we named HRXXN histidine kinases HhkA through HhkK. Phenotypic analysis of the mutants and complemented derivatives identified two functionally redundant kinases, HhkA and HhkE, that are required for nodulation competitiveness and during initiation of symbiosis. Using σEcfG-activity reporter strains, we further showed that both HhkA and HhkE activate the GSR in free-living cells exposed to salt and hyperosmotic stress. In conclusion, our data suggest that HhkA and HhkE trigger GSR activation in response to osmotically stressful conditions which B. diazoefficiens encounters during soybean host infection.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Ampicillin Treatment of Intracellular Listeria monocytogenes Triggers Formation of Persistent, Drug-Resistant L-Form Cells.

Listeria monocytogenes is an opportunistic intracellular pathogen causing an infection termed listeriosis. Despite the low incidence of listeriosis, the high mortality rate in individuals at risk makes this bacterium one of the most dangerous foodborne pathogens. Reports about a relapse of infection after antibiotic treatment suggest that the bacteria may be able to evade antibiotic treatment and persist as a dormant, antibiotic-tolerant subpopulation. In this study, we observed intracellular generation of antibiotic-resistant L-forms of Listeria monocytogenes following Ampicillin treatment of Listeria monocytogenes infected cells. Detection and identification of intracellular Listeria L-forms was performed by a combination of fluorescence in-situ hybridization and confocal laser scanning microscopy. Using micromanipulation, it was possible to isolate single intracellular L-form cells that following transfer into fresh medium gave rise to pure cultures. In conclusion, the results obtained here provide strong evidence that antibiotic treatment of infected host cells can induce the formation of L-forms from intracellular Listeria monocytogenes. Furthermore, our results suggest that intracellular L-forms persist inside host cells and that they represent viable bacteria, which are still able to grow and proliferate.

Engineering Endosymbiotic Growth of E. coli in Mammalian Cells.

Endosymbioses are cellular mergers in which one cell lives within another cell and have led to major evolutionary transitions, most prominently to eukaryogenesis. Generation of synthetic endosymbioses aims to provide a defined starting point for studying fundamental processes in emerging endosymbiotic systems and enable the engineering of cells with novel properties. Here, we tested the potential of different bacteria for artificial endosymbiosis in mammalian cells. To this end, we adopted the fluidic force microscopy technology to inject diverse bacteria directly into the cytosol of HeLa cells and examined the endosymbiont-host interactions by real-time fluorescence microscopy. Among them, Escherichia coli grew exponentially within the cytoplasm, however, at a faster pace than its host cell. To slow down the intracellular growth of E. coli, we introduced auxotrophies in E. coli and demonstrated that the intracellular growth rate can be reduced by limiting the uptake of aromatic amino acids. In consequence, the survival of the endosymbiont-host pair was prolonged. The presented experimental framework enables studying endosymbiotic candidate systems at high temporal resolution and at the single cell level. Our work represents a starting point for engineering a stable, vertically inherited endosymbiosis.

Promiscuous Installation of d-Amino Acids in Gene-Encoded Peptides.

d-Amino acids can have major effects on the structure, proteolytic stability, and bioactivity of peptides. Proteusin radical S-adenosyl methionine epimerases regioselectively install such residues in ribosomal peptides to generate peptides with the largest number of d-residues currently known in biomolecules. To study their utility in synthetic biology, we investigated the substrate tolerance and substrate-product relationships of the cyanobacterial model epimerase OspD using libraries of point mutants as well as distinct extended peptides that were fused to an N-terminal leader sequence. OspD was found to exhibit exceptional substrate promiscuity in E. coli, accepting 15 different amino acids and converting peptides with a broad range of compositions, secondary structures, and polarities. Diverse single and multiple epimerization patterns were identified that were dictated by the peptide sequence. The data suggest major potential in creating genetically encoded products previously inaccessible by synthetic biology.