# Subproject 3: Unique Features of Enterococcus that Confer Intrinsic Resistance

> **NIH NIH P01** · MASSACHUSETTS EYE AND EAR INFIRMARY · 2022 · $411,232

## Abstract

SUMMARY
The enterococci are ancient microbes that appear to have co-evolved along with the spread of
animals on land. They possess an intrinsic ruggedness that allows them to survive unusually
harsh conditions, including starvation and desiccation, yet efficiently recolonize the gut of a new
host. These traits distinguish them from their ancestors, which led to the identification of an initial
set of enterococcal-specific genes and phenotypes. Functional analysis by Tn-seq recently
implicated a subset of those genes is important for growth under non-challenged conditions, with
still others assuming importance upon antibiotic challenge. This subproject proposes to take
advantage of the power of high throughput single cell imaging to more rigorously identify genes
critical to the hardiness of enterococci, and associate them with precisely defined growth defects.
Moreover it expands this to include genes of importance to various probes of the cell wall,
including innate defense molecules, and in the process it will generate an ordered array of known
transposon insertion mutations in these genes of importance. As most of the genes that encode
the distinctive ruggedness of enterococci encode proteins of unknown function, these mutants
will be of considerable value for discovering the underlying mechanisms. Genes found to be
important are bioinformatically organized into putative functional networks, which create models
that can be tested experimentally. One such network emerged from preliminary studies, and
incorporates functions of three critical enterococcal genes with limited distribution outside of the
species, that encode genes of unknown function. Bioinformatic analysis suggests that these
genes play important roles in regulation of cell wall homeostasis, and appear to be influenced by
a quorum sensing mechanism. From functional analysis, this network appears to be highly
sensitive to the influence of rifampicin, supporting the role of several key inferred transcription
factors in the network. This network will be defined by creating tightly controlled gene silencing
constructs and identifying elements of the network through comparative transcriptomics and
proteomics. Because of the apparent involvement of three unique enterococcal genes in this
network, it appears to be a promising target for limiting the growth and survival of enterococci in
patients or in the hospital environment. It is expected that new insights gleaned from the applying
Mother Machine imaging technology will add new components to these networks with rigorously
defined contributions to enterococcal cell fitness.

## Key facts

- **NIH application ID:** 10327904
- **Project number:** 2P01AI083214-14
- **Recipient organization:** MASSACHUSETTS EYE AND EAR INFIRMARY
- **Principal Investigator:** Michael S Gilmore
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $411,232
- **Award type:** 2
- **Project period:** 2009-09-01 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10327904

## Citation

> US National Institutes of Health, RePORTER application 10327904, Subproject 3: Unique Features of Enterococcus that Confer Intrinsic Resistance (2P01AI083214-14). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10327904. Licensed CC0.

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