# Mechanism and function of membrane homeostasis of sortase modulated by an evolutionarily conserved protein involved in pilus assembly in Gram-positive bacteria

> **NIH NIH F31** · UNIVERSITY OF CALIFORNIA LOS ANGELES · 2021 · $15,741

## Abstract

Gram-positive bacteria assemble a unique class of covalently-linked protein polymers known as sortase-assembled pili or fimbriae that are important for polymicrobial interactions (or coaggregation), adhesion, and
bacterial virulence. Sortase-mediated pilus assembly is a two-step process: polymerization of pilus proteins
catalyzed by pilus-specific sortase is followed by cell wall anchoring of pilus polymers catalyzed by the
housekeeping sortase. How these sortases coordinate their enzymatic activities to assemble pili with an
optimal length on the cell surface is not well understood. In the oral bacterium Actinomyces oris, a key
colonizer in the development of oral biofilms, type 2 fimbriae, made of the fimbrial shaft FimA and the tip
coaggregation factor CafA, are essential for Actinomyces coaggregation with oral streptococci. The pilus-specific sortase SrtC2 polymerizes fimbrial polymers that are then anchored to the cell wall by the
housekeeping sortase SrtA. Importantly, mutant cells lacking srtA produce exceedingly long pili but fail to
mediate bacterial coaggregation. To elucidate a mechanism of SrtA modulation of pilus length, we sought to
characterize safA, a gene immediately downstream of srtA, since functionally related genes tend to cluster in
the bacterial genome. Remarkably, deletion of safA causes altered cell morphology, abolishment of
polymicrobial interactions, and production of exceedingly long pili, all phenotypes consistent with deletion of
srtA. These defective phenotypes were rescued with ectopic expression of safA from Corynebacterium
diphtheriae and Corynebacterium matruchotii in the A. oris safA mutant, suggesting a common mechanism in
Actinobacteria. Bacterial two-hybrid analysis demonstrated that SafA interacts with the housekeeping sortase
SrtA. Intriguingly, in the oral bacterium Bifidobacterium dentium, the housekeeping sortase SrtE contains a
fused homologous SafA sequence at its C-terminus. Altogether, it is hypothesized that SafA maintains
membrane homeostasis of SrtA through an evolutionarily conserved mechanism across Actinobacteria to
modulate pilus assembly and pilus length. Three specific aims are proposed to test this hypothesis. By genetic
and biochemical approaches, we aim to elucidate the mechanism of SrtA membrane homeostasis by the
evolutionary conserved protein SafA (Aim 1) and investigate the conservation of SafA and its co-evolution with
the housekeeping sortase SrtA (Aim 2). Using nematode and guinea pig models of infection, we aim to
examine the role of SafA in bacterial virulence (Aim 3). Besides providing a predoctoral training vehicle, this
proposed study will not only advance our understanding of surface assembly of bacterial virulence factors, but
also may provide attractive targets for anti-virulence strategies.

## Key facts

- **NIH application ID:** 10387439
- **Project number:** 1F31DE031500-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA LOS ANGELES
- **Principal Investigator:** Nicholas Anthony Ramirez
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $15,741
- **Award type:** 1
- **Project period:** 2022-04-01 → 2022-06-10

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10387439

## Citation

> US National Institutes of Health, RePORTER application 10387439, Mechanism and function of membrane homeostasis of sortase modulated by an evolutionarily conserved protein involved in pilus assembly in Gram-positive bacteria (1F31DE031500-01). Retrieved via AI Analytics 2026-08-11 from https://api.ai-analytics.org/grant/nih/10387439. Licensed CC0.

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