# Engineering of Complex Infectious Loci in Culture

> **NIH NIH R21** · TUFTS UNIVERSITY BOSTON · 2020 · $241,650

## Abstract

A wide swath of bacterial pathogens grow within deep tissue sites during disease. Pathogen growth
in these sites results in the recruitment of immune cells that attempt to clear of the invader, but these cells
are often ineffective because the virulent organism blocks the clearing process. As a consequence, the
microorganism sets up a beachhead where it can either establish a persistent infection or venture to spread
throughout the host. Depending on the nature of the recruited cells and tissue damage that occurs, these
foci of infection are referred to as abscesses, microabscesses, granulomas, or some combination of
processes. For many pathogens that grow outside of host cells, distinct microcolonies are formed, predicted
to result in considerable intermicrobial communication and direct targeting of host cells surrounding the
colony. An overriding problem in the infectious disease field is that the resulting architecture can only be
established in animal infection models and cannot be maintained or analyzed in culture. This work
proposes to overcome this stumbling block.
 Yersinia pseudotuberculosis is an enteropathogenic bacterium that can spread from the intestine
into regional lymph nodes, the liver and the spleen, establishing microcolonies surrounded by layers of
neutrophils, macrophages and inflammatory monocytes. The bacterium directly inactivates nearby
neutrophils, but there is a compensating attack by distal macrophages that generates nitric oxide (NO) and
its antimicrobial derivatives. Bacteria on the periphery of the microcolony inactivate NO, protecting their
centrally localized kin from exposure to toxic metabolites. The proposed Research Plan will exploit a
bioengineered gel microdroplet system to accurately reconstruct this battle. The Aims propose to analyze
Y. pseudotuberculosis interaction with immune cells, by growing bacteria in microcolonies within the gel
droplets, surrounding the droplets with adherent activated macrophages, morphologically mimicking a true
infectious site. Using a fluorescent reporter readout, the transcriptional profiles of peripheral and centrally
located bacteria will be analyzed, and compared to bacteria growing either in the absence of macrophage
stress or in a nonstructured environment. The system will be used to identify bacterial transcriptional circuits
that allow peripheral bacteria to maintain viability, and which protect the centrally located kin from attack. It
will also identify the bacterial transcriptional response to growth in aggregates found in tissues, as well as
identify previously uncharacterized physiological and stress responses of the small bacterial community to
secreted macrophage products. Successful completion of the Aims is part of the long-term goal of
determining how inter-bacterial interactions ensure the establishment of an infectious niche, and to evaluate
how anti-microbial immune cells collaborate with pathogens to promote disease.

## Key facts

- **NIH application ID:** 9955709
- **Project number:** 1R21AI151593-01
- **Recipient organization:** TUFTS UNIVERSITY BOSTON
- **Principal Investigator:** Ralph R. Isberg
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $241,650
- **Award type:** 1
- **Project period:** 2020-02-01 → 2022-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9955709

## Citation

> US National Institutes of Health, RePORTER application 9955709, Engineering of Complex Infectious Loci in Culture (1R21AI151593-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9955709. Licensed CC0.

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