# Copper tolerance and homeostasis in Pneumocystis species

> **NIH NIH R21** · CINCINNATI CHILDRENS HOSP MED CTR · 2020 · $199,913

## Abstract

Abstract
All organisms must maintain a balance between acquiring sufficient amounts of essential trace metals for
growth and proliferation, and the toxicity associated with their elevated levels. Mammalian hosts use both
copper limitation and copper overload as defense mechanisms against fungal pathogens. Fungi from the
Pneumocystis genus cause pneumonia (PCP) in mammals with weakened immune systems. Mammalian
lungs are a high copper environment, and these fungi maintain an extracellular life cycle in the lung alveoli
which brings them into direct contact with an abundance of host factors including metals. In this pilot project,
we seek to characterize molecular mechanisms underlying copper tolerance and homeostasis in
Pneumocystis murina (Pm) that enable the fungus to counter the host Cu-driven cytotoxicity. The trace
metal content was measured in Pm samples freshly extracted from mouse lungs with PCP infection, using
size-exclusion chromatography (SEC). From a wide panel of measured metals, only Cu-, Fe-, and Zn-binding
proteins of Pm showed pronounced distinct chromatographic profiles with elevated levels after accounting for
the mouse metalloproteome background. Of the essential metals, copper is unique as it is required in trace
amounts but quickly becomes highly toxic at elevated levels. The role of Cu in Pneumocystis biology is not
known. Understanding Pm molecular mechanisms involved in Cu homeostasis will shed light on adaptation
strategies of the fungus and may reveal new virulence factors and potential drug targets. The following
specific aims are proposed: Aim 1. Identification of Cu-binding proteins – to reveal (i) the Cu proteome of
the host lungs infected with Pm and (ii) the primary Cu-binding proteins of the pathogen under pressure of
excessive labile copper cytotoxicity. Cu-binding proteins will be separated and quantified using SEC-ICP-MS.
Protein fractions from Pm and lung tissue will be enriched for Cu-proteins by immobilized metal-ion affinity
chromatography (IMAC). Isolated proteins will be identified using shotgun proteomics (LC-MS/MS). Aim 2.
Identification of Pm proteins induced by labile copper – to identify the functional genes of the pathogen
that orchestrate responses to excessive extracellular Cu. Gene induction by labile Cu2+ will be measured
using RNA-seq following the addition of CuSO4 to the media using two concentrations, 1ng/ml and 100ng/ml,
at 4-time points to reveal dynamic changes in gene expression. The project will advance knowledge on the
Pneumocystis biology (Cu homeostasis) and shed light on the new axis of host-pathogen interactions for
extracellular pathogens.

## Key facts

- **NIH application ID:** 9971459
- **Project number:** 5R21AI143467-02
- **Recipient organization:** CINCINNATI CHILDRENS HOSP MED CTR
- **Principal Investigator:** Alexey Porollo
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $199,913
- **Award type:** 5
- **Project period:** 2019-07-05 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9971459

## Citation

> US National Institutes of Health, RePORTER application 9971459, Copper tolerance and homeostasis in Pneumocystis species (5R21AI143467-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9971459. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*