Genomic profiling and experimental validation of type VI secretion system-associated proteins in Klebsiella
Ning Zhu, Yuzhe Zhao, Wenjing Yi, Lili Jiang, Tingting Dong, Zhihan Peng, Shanshan Jia, Ruihong Wu, Xiaohan Guo, Arnab Pain, Lei Song, Qingtian Guan
Abstract
Klebsiella is widely distributed in nature and recognized for its dual role as a human commensal and an opportunistic pathogen capable of causing severe infections. Among its key virulence mechanisms, the Type VI Secretion System (T6SS) plays a critical role in microbial competition, interspecies interactions, and environmental adaptation. In this study, we investigated a representative set of high-quality Klebsiella genomes and identified two major T6SS loci (T6SSkleb1 and T6SSkleb2), which exhibit distinct structural features and are predominantly found within the Klebsiella pneumoniae species complex (KpSC).
Introduction
The genus Klebsiella, a group of Gram-negative, oxidase-negative, rod-shaped bacteria, that are normally found as normal flora in the nose, mouth, and gastrointestinal tract; however, they can also act as opportunistic human pathogens. Klebsiella organisms can cause various disease states, notably pneumonia, urinary tract infections, sepsis, meningitis, diarrhea, peritonitis and soft tissue infections [1]. Among species of the genus Klebsiella, K. pneumoniae is the most prevalent.
Materials and method
Data acquisition and quality assessment
The comprehensive assemblies from Klebsiella genus available in the NCBI Genome database (as of November.1th, 2023) were downloaded. We first performed a rarefaction analysis to evaluate the diversity of T6SS-associated proteins. The rarefaction curve demonstrated that the number of unique T6SS-associated proteins, including effectors, immunity proteins and regulators rapidly approached saturation as the number of genomes increased (S6 Fig).
Results
Identification and characterization of T6SS loci and phylogeny analysis in Klebsiella spp.
The high-quality genome that passed the quality control (S1 Table) were compared against the protein database obtained from SecReT6 [25] using Proteinortho v 6.3.0 [26] to identify T6SS loci based on the T6SS core components and other regionally intrinsic proteins. The distribution of T6SS of total 4,434 genomes from the NCBI databases was evaluated and two major types of T6SS loci in Klebsiella genus have been identified, here, namely, T6SSkleb1 and T6SSkleb2, respectively.
Discussion
In this study, we conducted a comprehensive analysis of the T6SS including core structural components, effectors and immunity proteins in Klebsiella genus. We investigated the genes families that are significantly enriched in the presence of T6SS, and validate novel putative effectors in Klebsiella spp.. We found that majority of Klebsiella strains possess a complete T6SS apparatus, organized into two distinct loci, designated as T6SSkleb1 and T6SSkleb2. Similar locus architectures have previously been identified in specific strains, such as K.
Acknowledgments
We extend our sincere thanks to the colleagues from Bioinformatics Platform and Infectious Diseases and Pathogen Biology Center at The First Hospital of Jilin University for their valuable discussions and suggestions, which have significantly enhanced the quality of this manuscript.
Citation: Zhu N, Zhao Y, Yi W, Jiang L, Dong T, Peng Z, et al. (2025) Genomic profiling and experimental validation of type VI secretion system-associated proteins in Klebsiella. PLoS Genet 21(9): e1011878. https://doi.org/10.1371/journal.pgen.1011878
Editor: Diarmaid Hughes, Uppsala University: Uppsala Universitet, SWEDEN
Received: April 24, 2025; Accepted: September 10, 2025; Published: September 19, 2025
Copyright: © 2025 Zhu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The genome assembly summary and the phylogenetic tree files used in this study are available at Zenodo (https://doi.org/10.5281/zenodo.17044063).
Funding: This work was supported by the Jilin Provincial Science and Technology Development Project (YDZJ202401276ZYTS to Q.G.), the Bethune Project of Jilin University (2024B20 to L.S.), and the Science and Technology Development Project of Changchun City (23YQ10 to L.S.). No authors received salary support from these funders.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
