A combination of genomics and transcriptomics provides insights into the distribution and differential mRNA expression of type VI secretion system in clinical Klebsiella pneumoniae

Abstract

The type VI secretion system (T6SS) serves as a crucial molecular weapon in interbacterial competition and significantly influences the adaptability of bacteria in their ecological niche. However, the distribution and function of T6SS in clinical Klebsiella pneumoniae, a common opportunistic nosocomial pathogen, have not been fully elucidated. Here, we conducted a genomic analysis of 65 clinical K. pneumoniae isolates obtained from patients with varying infections. Genes encoding a T6SS cluster present in all analyzed strains of K. pneumoniae, and strains with identical sequence type carried structurally and numerically identical T6SS. Our study also highlights the importance of selecting conserved regions within essential T6SS genes for PCR-based identification of T6SS in bacteria. Afterward, we utilized the predominant sequence type 11 (ST11) K. pneumoniae HS11286 to investigate the effect of knocking out T6SS marker genes hcp or vgrG. Transcriptome analysis identified a total of 1,298 co-upregulated and 1,752 co-downregulated differentially expressed genes in both mutants. Pathway analysis showed that only Δhcp mutant exhibited alterations in transport, establishment of localization, localization, and cell processes. The absence of hcp or vgrG gene suppressed the expression of other T6SS-related genes within the locus I cluster. Additionally, interbacterial competition experiments showed that hcp and vgrG are essential for competitive ability of ST11 K. pneumoniae HS11286. This study furthers our understanding of the genomic characteristics of T6SS in clinical K. pneumoniae and suggests the involvement of multiple genes in T6SS of strain HS11286.

Importance: Gram-negative bacteria use type VI secretion system (T6SS) to deliver effectors that interact with neighboring cells for niche advantage. Klebsiella pneumoniae is an opportunistic nosocomial pathogen that often carries multiple T6SS loci, the function of which has not yet been elucidated. We performed a genomic analysis of 65 clinical K. pneumoniae strains isolated from various sources, confirming that all strains contained T6SS. We then used transcriptomics to further study changes in gene expression and its effect on interbacterial competition following the knockout of key T6SS genes in sequence type 11 (ST11) K. pneumoniae HS11286. Our findings revealed the distribution and genomic characteristics of T6SS in clinical K. pneumoniae. This study also described the overall transcriptional changes in the predominant Chinese ST11 strain HS11286 upon deletion of crucial T6SS genes. Additionally, this work provides a reference for future research on the identification of T6SS in bacteria.

Keywords: Klebsiella pneumoniae; genomic; transcriptomic; type VI secretion system.

Fig 1

Phylogenetic tree of 66 clinical K. pneumoniae strains. The different colors represent different ST types. Column 1 shows the type of infection and column 2 the CPS loci. In column 3, a colored-filled square indicates the collection date. Square-colored blue represents the presence of the T6SS-related genes labeled above the figure. BSI: bloodstream infection; IAI: intra-abdominal infection; HAP: hospital-acquired pneumonia; CNSI: central nervous system infection; LA: liver abscess; and UNK: unknown.

Fig 2

Schematic representation of T6SS clusters in different sequence types of K. pneumoniae. ST11 K. pneumoniae was represented by strain HS11286, which possesses two copies of T6SS. Sequence type 23 (ST23) K. pneumoniae was represented by strain NTUH-K2044, which also possesses two copies of T6SS. Sequence type 15 (ST15) of K. pneumoniae contains three copies of T6SS, with strain KP17-16 serving as an example.

Fig 3

Comparative analysis of the type i2 T6SS cluster in different sequence types of K. pneumoniae. (A) The genetic environments of three T6SSs obtained from ST23 NTUH-K2044, ST11 HS11286, and ST15 KP17-16 strains are presented as a linear alignment. T6SS-related genes are represented in colored arrows. Bidirectional BLAST hits were illustrated to denote sequence identity ranging from 66% to 100%. (B) A comparative gene analysis of hcp, vgrG, and tssM among different sequence types of K. pneumoniae strains.

Fig 4

Comparative analyses of the transcriptional response in Δhcp and ΔvgrG K. pneumoniae mutants. (A) Venn diagrams showing the number of significantly upregulated or downregulated genes (DEGs; |log₂FoldChange| > 1, padj < 0.05) in the different K. pneumoniae mutants compared to the WT. (B) Gene ontology analysis of up-regulated and down-regulated DEGs in Δhcp/WT. CC: cellular component and MF: molecular function. (C) Differential expression of T6SS clusters in Δhcp and ΔvgrG K. pneumoniae mutants.

Fig 5

Interspecies killing by K. pneumoniae HS11286 in a contact-dependent manner. (A–C) Interbacterial competitive growth assays. Surviving E. coli EC600 after 24 h of coincubation with (A) HS11286 (WT) and the Δhcp and ΔvgrG mutants; (B) the WT containing the empty vector (WT/pHSG398), the Δhcp mutant containing the empty vector (Δhcp/pHSG398), and the Δhcp mutant complemented with pHSG398-hcp (Δhcp/hcp); (C) WT/pHSG398, the ΔvgrG mutant harboring pHSG398 (ΔvgrG/pHSG398), and the ΔvgrG mutant harboring pHSG398-hcp (ΔvgrG/vgrG). A control lacking any K. pneumoniae strain (none) was also included. Recovered mixtures were plated onto Luria-Bertani agar supplemented with 100 µg/mL rifampicin. (D) Survival rates of prey strain EC600 following 24 h of coincubation with the associated predator strain (K. pneumoniae HS11286 [WT], Δhcp mutant, ΔvgrG mutant, hcp and vgrG complemented strains). The data represent the means of three independent trials. *P < 0.05 by one-way analysis of variance (Δhcp mutant or ΔvgrG mutant compared with the WT strain).