# In Situ Architecture of Specialized Bacterial Secretion Systems

> **NIH NIH R35** · UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON · 2020 · $390,000

## Abstract

Bacteria have evolved specialized nanomachines functioning as secretion systems to deliver proteins or DNA
from the bacterial cytoplasm to the surrounding milieu or into other eukaryotic or bacterial target cells. To
date, nine different types of bacterial secretion systems have been identified. The most widely-distributed and
versatile of these, the type IV secretion systems (T4SSs), traverse the cell envelopes of many Gram-negative
and -positive species. Members of one large subfamily, the DNA transfer or conjugation systems, are
medically problematic because they deliver mobile genetic elements (MGEs) and their cargoes of antibiotic
resistance genes and virulence determinants among bacterial populations; these systems also elaborate
conjugative pili or other surface adhesins that promote establishment of robust, antibiotic-resistant biofilm
communities. A second T4SS subfamily, the ‘effector translocators’ are deployed by many medically-
important pathogens to deliver protein effectors across the cell envelope either to the surrounding milieu or
into eukaryotic host cells to incite infection. By use of in situ cryo-electron tomography (Cryo-ET), I have
recently solved the structures of three different T4SSs, the Legionella pneumophila Dot/Icm, Escherichia coli
F plasmid Tra, and Helicobacter pylori Cag systems within their natural cell envelopes. These new structures
are changing existing paradigms for how T4SSs are architecturally configured, they present the first clear
views of central substrate translocation channels, and they identify novel F-encoded structures configured as
basal platforms for F pili. Type IX secretion systems (T9SSs), which are found mainly in the phylum
Bacteroidetes, also play critical roles in infection. Porphyromonas gingivalis, for example, deploys its T9SS to
secrete gingipain proteinases and virulence factors to incite periodontal disease. Very recently, I solved the
structure of this T9SS in its natural cellular context by in situ Cryo-ET. This large (~50 nm diameter),
envelope-spanning nanomachine differs markedly from any other bacterial secretion systems visualized to
date. In this MIRA proposal, I seek to comprehensively define the structures and subunit compositions of the
F plasmid Tra and H. pylori Cag T4SSs and the P. gingivalis T9SS by addressing key unresolved questions
that are ideally or uniquely approachable using in situ Cryo-ET. We will i) solve in situ structures with
emphasis on regions of these nanomachines such as the inner membrane complexes, translocation channels,
and machine - pilus junctions that have not been amenable to structural analyses using in vitro approaches, ii)
leverage our resources through collaborations with experts in the T4SS and T9SS fields to place our
structural findings in broader mechanistic and biological contexts, and iii) refine methods for data collection
and processing to improve the resolution limits of in situ Cryo-ET. Our studies will generate important new
in...

## Key facts

- **NIH application ID:** 10028548
- **Project number:** 1R35GM138301-01
- **Recipient organization:** UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
- **Principal Investigator:** Bo Hu
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $390,000
- **Award type:** 1
- **Project period:** 2020-09-04 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10028548

## Citation

> US National Institutes of Health, RePORTER application 10028548, In Situ Architecture of Specialized Bacterial Secretion Systems (1R35GM138301-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10028548. Licensed CC0.

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