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Background <p>Opportunistic pathogens such as <i>Klebsiella pneumoniae</i> and <i>Acanthamoeba castellanii</i> frequently coexist in environmental water bodies, where their interactions can influence bacterial pathogenicity. Our previous work demonstrated that <i>A. castellanii</i> genotype T4 (AcT4) can induce capsule enlargement in <i>K. pneumoniae</i>, suggesting that it is a potential environmental source of hypervirulent strains. However, the molecular mechanisms underlying this transformation remain unclear.</p> Methods <p>We investigated the effects of AcT4 on the capsule formation and virulence of <i>K. pneumoniae</i> using co-culture assays, antibiotic susceptibility testing, lactate dehydrogenase (LDH) cytotoxicity assays, and serum resistance assays. Capsule-associated gene expression (<i>wzi</i>, <i>galF</i>, <i>rcsB</i>, <i>rmpA</i>, and <i>rmpA2</i>) was analysed via reverse transcription–quantitative polymerase chain reaction (RT-qPCR). Excretory–secretory proteins (ESPs) from AcT4 were separated into &gt;10&#xa0;kDa and &lt;10&#xa0;kDa fractions to identify active components. Ion concentrations in amoeba-conditioned media were measured to assess environmental stress factors.</p> Results <p>Co-culture with AcT4 significantly increased <i>K. pneumoniae</i> cytotoxicity toward A549 cells and enhanced serum resistance without altering antibiotic susceptibility. RT-qPCR revealed significant upregulation of <i>rcsB</i>, <i>galF</i>, and <i>wzi</i> in the induced strains, while the expression of the virulence plasmid genes <i>rmpA/rmpA2</i> was absent. Amoeba-conditioned media altered ion distributions, notably increasing iron levels, and ESP fractionation revealed &lt;10&#xa0;kDa molecules as the primary drivers of capsule enlargement and <i>wzi</i> upregulation.</p> Conclusions <p>Small (&lt;10&#xa0;kDa) ESPs from AcT4 can induce capsule-associated gene expression in <i>K. pneumoniae</i> via activation of the Rcs two-component system, independent of virulence plasmids. These findings reveal a novel contact-independent mechanism by which environmental protozoa may contribute to the emergence of hypervirulent <i>K. pneumoniae</i>, highlighting potential public health risks from aquatic environments.</p> Graphical Abstract <p></p>

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Small excretory–secretory proteins of Acanthamoeba castellanii genotype T4 upregulate the expression of capsule-associated genes via the Rcs two-component system in Klebsiella pneumoniae

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  • 发表日期：

    2025年11月19日

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  • Yen-Chu Chuang,
  • Hsin-Yu Hsieh,
  • Chih-Ming Tsai,
  • Yu-Jen Wang

摘要

Background

Opportunistic pathogens such as Klebsiella pneumoniae and Acanthamoeba castellanii frequently coexist in environmental water bodies, where their interactions can influence bacterial pathogenicity. Our previous work demonstrated that A. castellanii genotype T4 (AcT4) can induce capsule enlargement in K. pneumoniae, suggesting that it is a potential environmental source of hypervirulent strains. However, the molecular mechanisms underlying this transformation remain unclear.

Methods

We investigated the effects of AcT4 on the capsule formation and virulence of K. pneumoniae using co-culture assays, antibiotic susceptibility testing, lactate dehydrogenase (LDH) cytotoxicity assays, and serum resistance assays. Capsule-associated gene expression (wzi, galF, rcsB, rmpA, and rmpA2) was analysed via reverse transcription–quantitative polymerase chain reaction (RT-qPCR). Excretory–secretory proteins (ESPs) from AcT4 were separated into >10 kDa and <10 kDa fractions to identify active components. Ion concentrations in amoeba-conditioned media were measured to assess environmental stress factors.

Results

Co-culture with AcT4 significantly increased K. pneumoniae cytotoxicity toward A549 cells and enhanced serum resistance without altering antibiotic susceptibility. RT-qPCR revealed significant upregulation of rcsB, galF, and wzi in the induced strains, while the expression of the virulence plasmid genes rmpA/rmpA2 was absent. Amoeba-conditioned media altered ion distributions, notably increasing iron levels, and ESP fractionation revealed <10 kDa molecules as the primary drivers of capsule enlargement and wzi upregulation.

Conclusions

Small (<10 kDa) ESPs from AcT4 can induce capsule-associated gene expression in K. pneumoniae via activation of the Rcs two-component system, independent of virulence plasmids. These findings reveal a novel contact-independent mechanism by which environmental protozoa may contribute to the emergence of hypervirulent K. pneumoniae, highlighting potential public health risks from aquatic environments.

Graphical Abstract