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. 2023 Sep 21;11(5):e0117023.
doi: 10.1128/spectrum.01170-23. Online ahead of print.

The role of integration host factor in biofilm and virulence of high-alcohol-producing Klebsiella pneumoniae

Zheng Fan #  1 , Tongtong Fu #  1 , Zhoufei Li #  1   2 , Bing Du  3 , Xiaohu Cui  1 , Rui Zhang  1   2 , Yanling Feng  1 , Hanqing Zhao  1 , Guanhua Xue  1 , Jinghua Cui  1 , Chao Yan  1 , Lin Gan  1 , Junxia Feng  1 , Ziying Xu  1 , Zihui Yu  1 , Ziyan Tian  1 , Zanbo Ding  1 , Jinfeng Chen  1 , Yujie Chen  1 , Jing Yuan  1
Affiliations

The role of integration host factor in biofilm and virulence of high-alcohol-producing Klebsiella pneumoniae

Zheng Fan et al. Microbiol Spectr. .

Abstract

Klebsiella pneumoniae is a well-known human nosocomial pathogen with an arsenal of virulence factors, including capsular polysaccharides (CPS), fimbriae, flagella, and lipopolysaccharides (LPS). Our previous study found that alcohol acted as an essential virulence factor for high-alcohol-producing K. pneumoniae (HiAlc Kpn). Integration host factor (IHF) is a nucleoid-associated protein that functions as a global virulence regulator in Escherichia coli. However, the regulatory role of IHF in K. pneumoniae remains unknown. In the present study, we found that deletion of ihfA or ihfB resulted in a slight defect in bacterial growth, a severe absence of biofilm formation and cytotoxicity, and a significant reduction in alcohol production. RNA sequencing differential gene expression analysis showed that compared with the wild-type control, the expression of many virulence factor genes was downregulated in ΔihfA and ΔihfB strains, such as those related to CPS (rcsA, galF, wzi, and iscR), LPS (rfbABCD), type I and type III fimbriae (fim and mrk operon), cellulose (bcs operon), iron transporter (feoABC, fhuA, fhuF, tonB, exbB, and exbD), quorum sensing (lsr operon and sdiA), type II secretion system (T2SS) and type VI secretion system (T6SS) (tssG, hcp, and gspE). Of these virulence factors, CPS, LPS, fimbriae, and cellulose are involved in biofilm formation. In addition, IHF could affect the alcohol production by regulating genes related to glucose intake (ptsG), pyruvate formate-lyase, alcohol dehydrogenase, and the tricarboxylic acid (TCA) cycle. Our data provided new insights into the importance of IHF in regulating the virulence of HiAlc Kpn. IMPORTANCE Klebsiella pneumoniae is a well-known human nosocomial pathogen that causes various infectious diseases, including urinary tract infections, hospital-acquired pneumonia, bacteremia, and liver abscesses. Our previous studies demonstrated that HiAlc Kpn mediated the development of nonalcoholic fatty liver disease by producing excess endogenous alcohol in vivo. However, the regulators regulating the expression of genes related to metabolism, biofilm formation, and virulence of HiAlc Kpn remain unclear. In this study, the regulator IHF was found to positively regulate biofilm formation and many virulence factors including CPS, LPS, type I and type III fimbriae, cellulose, iron transporter, AI-2 quorum sensing, T2SS, and T6SS in HiAlc Kpn. Furthermore, IHF positively regulated alcohol production in HiAlc Kpn. Our results suggested that IHF could be a potential drug target for treating various infectious diseases caused by K. pneumoniae. Hence, the regulation of different virulence factors by IHF in K. pneumoniae requires further investigation.

Keywords: HiAlc Kpn; IHF; biofilm; regulate; virulence.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Deletion of ihfA or ihfB slightly influenced the growth of HiAlc Kpn in LB broth. (A) The growth curves of W14, ΔihfA, ΔihfB, ΔihfA/ihfA, and ΔihfB/ihfB were measured by optical density (OD600) per hour over a period of 12 h. (B) Typical colony images of W14, ΔihfA, and ΔihfB.
FIG 2
FIG 2
Deletion of ihfA or ihfB decreased the biofilm formation and virulence in HiAlc Kpn. (A) Images of biofilm formed by W14, ΔihfA, ΔihfB, ΔihfA/ihfA, and ΔihfB/ihfB were stained with 1% CV and adhered on plastic centrifuge tubes. (B) Biofilm was stained with 1% CV. The extracted color was dissolved with 33% bleaching solution and measured at OD590. ***P < 0.001, compared to wild-type strain W14 by Student’s t-test. (C) CLSM of biofilm formation in W14, ΔihfA, ΔihfB, ΔihfA/ihfA, and ΔihfB/ihfB was observed after incubation for 48 h. (D) Cytotoxicity of W14, ΔihfA, ΔihfB, ΔihfA/ihfA, and ΔihfB/ihfB. A549 cells were infected with the indicated strains at a MOI of 100. After 10 h of infection and 15-min crystal violet staining, cells attached to the plate were measured at OD490. ***P < 0.001, compared to wild-type strain W14 by Student’s t-test.
Fig 3
Fig 3
Deletion of ihfA or ihfB decreased the alcohol production in HiAlc Kpn. The alcohol-producing ability of W14, ΔihfA, ΔihfB, ΔihfA/ihfA, and ΔihfB/ihfB was measured in aerobic and anaerobic conditions. ***P < 0.001, compared to wild-type strain W14 by Student’s t-test.
FIG 4
FIG 4
Profiling gene expression of ΔihfA and ΔihfB. Analysis of total RNA sequencing. (A) The Venn diagram shows the overlapped DEGs numbers of W14 and ΔihfA, W14 and ΔihfB, ΔihfA and ΔihfB. (B) Volcano plot of DEGs between W14 and ΔihfA or ΔihfB. GO enrichment analysis of upregulated (C) and downregulated (D) DEGs in ΔihfA and ΔihfB. KEGG pathway enrichment analysis of upregulated DEGs in ΔihfA (E) and ΔihfB (F). KEGG pathway enrichment analysis of downregulated DEGs in ΔihfA (G) and ΔihfB (H).
FIG 5
FIG 5
Regulatory network of IHF in HiAlc Kpn. The regulatory network of IHF in HiAlc Kpn. IHF positively regulates the synthesis of CPS, LPS, cellulose, type I and type III fimbriae, T2SS, T6SS, iron transporter and QS by IHF. Gene expression measured by RNA sequencing is shown by heat map.
FIG 6
FIG 6
Deletion of ihfA or ihfB decreased the expressions of biofilm and virulence-related genes. The relative mRNA levels of W14, ΔihfA, and ΔihfB in CPS (A), LPS (B), cellulose (C), type I fimbriae (D), type III fimbriae (E), QS system (F), iron acquisition (G), and other virulence-related genes (H) were calculated by qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001, compared to wild-type strain W14 by Student’s t-test.
FIG 7
FIG 7
IHF-regulated genes related to the TCA cycle and fermentation. (A) Metabolic pathways and related genes of HiAlc Kpn. The picture shows up- and downregulated genes in ΔihfA and ΔihfB compared with wild-type strain W14. (B) The relative mRNA levels of fermentation-related genes in W14, ΔihfA, and ΔihfB were calculated by qRT-PCR. (C) The alcohol-producing ability of W14, ΔihfA, ΔihfB, ΔihfA/adhE, and ΔihfB/adhE was measured in aerobic and anaerobic conditions. (D) The relative mRNA levels of TCA cycle-related genes in W14, ΔihfA, and ΔihfB were calculated by qRT-PCR. *P < 0.05; **P < 0.01, ***P < 0.001, compared to wild-type strain W14 by Student’s t-test.
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