high-resolution-three-dimensional-whole-organ-tomography-microbial

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...

University of Basel

Host control of infections crucially depends on the capability to kill pathogens with reactive oxygen species (ROS). However, these toxic molecules can also readily damage host components and cause severe immunopathology. Here, we show that neutrophils use their most abundant granule protein, myeloperoxidase, to target ROS specifically to pathogens while minimizing collateral tissue damage. A computational model predicted that myeloperoxidase efficiently scavenges diffusible HO at the surface of phagosomal Salmonella and converts it into highly reactive HOCl (bleach), which rapidly damages biomolecules within a radius of less than 0.1 μm. Myeloperoxidase-deficient neutrophils were predicted to accumulate large quantities of HO that still effectively kill Salmonella, but most HO would leak from the phagosome. Salmonella stimulation of neutrophils from normal and myeloperoxidase-deficient human donors experimentally confirmed an inverse relationship between myeloperoxidase activity and extracellular HO release. Myeloperoxidase-deficient mice infected with Salmonella had elevated hydrogen peroxide tissue levels and exacerbated oxidative damage of host lipids and DNA, despite almost normal Salmonella control. These data show that myeloperoxidase has a major function in mitigating collateral tissue damage during antimicrobial oxidative bursts, by converting diffusible long-lived HO into highly reactive, microbicidal and locally confined HOCl at pathogen surfaces.

Myeloperoxidase targets oxidative host attacks to Salmonella and prevents collateral tissue damage.

Antibiotic chemotherapy effectively cures many infections caused by susceptible bacterial pathogens. However, in some cases, even extended treatment duration does not completely eradicate the pathogenic bacteria from host tissues. A common model for underlying mechanisms assumes the stochastic formation of bacterial persisters similar to observations in laboratory cultures. However, alternative explanations related to the complexity of infected host tissues could also be relevant. We discuss several of these aspects and emphasize the need for integrated analysis as a basis for new control strategies.

Antibiotic chemotherapy against heterogeneous pathogen populations in complex host tissues.

Antimicrobial chemotherapy can fail to eradicate the pathogen, even in the absence of antimicrobial resistance. Persisting pathogens can subsequently cause relapsing diseases. In vitro studies suggest various mechanisms of antibiotic persistence, but their in vivo relevance remains unclear because of the difficulty of studying scarce pathogen survivors in complex host tissues. Here, we localized and characterized rare surviving  in mouse spleen using high-resolution whole-organ tomography. Chemotherapy cleared >99.5% of the  but was inefficient against a small  subset in the white pulp. Previous models could not explain these findings: drug exposure was adequate,  continued to replicate, and host stresses induced only limited  drug tolerance. Instead, antimicrobial clearance required support of -killing neutrophils and monocytes, and the density of such cells was lower in the white pulp than in other spleen compartments containing higher  loads. Neutrophil densities declined further during treatment in response to receding  loads, resulting in insufficient support for  clearance from the white pulp and eradication failure. However, adjunctive therapies sustaining inflammatory support enabled effective clearance. These results identify uneven  tissue colonization and spatiotemporal inflammation dynamics as main causes of  persistence and establish a powerful approach to investigate scarce but impactful pathogen subsets in complex host environments.

Tissue compartmentalization enables  persistence during chemotherapy.

Virulence and persistence of the obligate intracellular parasite Toxoplasma gondii involve the secretion of effector proteins belonging to the family of dense granule proteins (GRAs) that act notably as modulators of the host defense mechanisms and participate in cyst wall formation. The subset of GRAs residing in the parasitophorous vacuole (PV) or exported into the host cell, undergo proteolytic cleavage in the Golgi upon the action of the aspartyl protease 5 (ASP5). In tachyzoites, ASP5 substrates play central roles in the morphology of the PV and the export of effectors across the translocon complex MYR1/2/3. Here, we used N-terminal amine isotopic labeling of substrates to identify novel ASP5 cleavage products by comparing the N-terminome of wild-type and Δasp5 lines in tachyzoites and bradyzoites. Validated substrates reside within the PV or PVM in an ASP5-dependent manner. Remarkably, Δasp5 bradyzoites are impaired in the formation of the cyst wall in vitro and exhibit a considerably reduced cyst burden in chronically infected animals. More specifically two-photon serial tomography of infected mouse brains revealed a comparatively reduced number and size of the cysts throughout the establishment of persistence in the absence of ASP5.

Jiagui Li

Biozentrum

University of Basel

High-Resolution Three-Dimensional Whole-Organ Tomography of Microbial Infections