Novel Virulence Factors Deciphering Klebsiella pneumoniae KpC4 Infect Maize as a Crossing-Kingdom Pathogen: An Emerging Environmental Threat

Klebsiella pneumoniae is not only a human and animal opportunistic pathogen, but a food-borne pathogen. Cross-kingdom infection has been focused on since K. pneumoniae was identified as the pathogen of maize, banana, and pomegranate. Although the pathogenicity of K. pneumoniae strains (from ditch water, maize, and human) on plant and mice has been confirmed, there are no reports to explain the molecular mechanisms of the pathogen. This study uncovered the K. pneumoniae KpC4 isolated from maize top rot for the determination of various virulence genes and resistance genes. At least thirteen plant disease-causing genes are found to be involved in the disruption of plant defense. Among them, rcsB is responsible for causing disease in both plants and animals. The novel sequence types provide solid evidence that the pathogen invades plant and has robust ecological adaptability. It is imperative to perform further studies on the verification of these KpC4 genes’ functions to understand the molecular mechanisms involved in plant–pathogen interactions.     

代谢副产物对Klebsiella pneumoniae生长及合成1,3-丙二醇的影响

研究了Klebsiella pneumoniae在厌氧摇瓶中的生长代谢特性和基质消耗情况,发现主要副产物乙醇是抑制菌体持续生长及1,3-丙二醇合成的主要因素,外源添加实验表明,8 g/L乙醇可使K. pneumoniae比生长速率、1,3-丙二醇比合成速率、最大菌体浓度及1,3-丙二醇终浓度分别下降21.6%, 22.1%, 59.6%及33.5%;指数生长期加入乙醇对菌体生长代谢的抑制作用更加明显. 其他代谢副产物乙酸、乳酸、2,3-丁二醇对K. pneumoniae生长代谢也有不同程度影响,乙酸浓度仅2 g/L即可对菌体生长产生抑制,乙酸浓度达到5 g/     

3-羟丙醛对Klebsiella pneumoniae发酵产 1,3-丙二醇的影响及其调控

中间产物3-羟丙醛在发酵液中的积累对Klebsiella pneumoniae细胞生长及1,3-丙二醇的合成有显著的抑制作用,而调节发酵的起始甘油浓度及控制发酵pH值可调控发酵液中3-羟丙醛的积累.当起始甘油质量浓度分别为20、30、50、70g/L的批式发酵中,发酵液中3-羟丙醛的积累的高峰分别为4.31、6.87、11.48及13.49mmol/L,当起始甘油质量浓度大于50g/L时,3-羟丙醛在到达积累高峰后不能被菌体有效转化,在发酵后期维持较高浓度,抑制了细胞生长及1,3-丙二醇的合成,发酵不能继续进行.控制发酵pH值为7.75～8.0可促进发酵液堆积的3-羟丙醛被迅速转化.在流加发酵中起始甘油质量浓度采用30g/L,发酵pH值控制为7.75条件下,发酵32 h,1,3-丙二醇质量浓度可达37.16g/L,1,3-丙二醇的生产强度和质量得率分别达到1.16g/(L·h)和52.66%.     

蔗糖与葡萄糖作辅助碳源对重组Klebsiella pneumoniae发酵生产 1,3-丙二醇的影响

实验研究了重组Klebsiella pneumoniae批式发酵生产1,3-丙二醇过程中辅助碳源蔗糖与葡萄糖对发酵过程的影响,对发酵工艺进行了放大,并对流加策略进行了优化. 结果表明,葡萄糖为发酵生产1,3-丙二醇的辅助碳源优于蔗糖;以重组Klebsiella pneumoniae为菌种,以葡萄糖为辅助碳源,采用指数流加策略,30 L发酵罐中1,3-丙二醇的产量最高达85.2 g/L,产率达0.63 mol/mol,比单纯以甘油为碳源分别提高37.35%和25.00%.     

The Therapeutic Treatment with the GAG-Binding Chemokine Fragment CXCL9(74–103) Attenuates Neutrophilic Inflammation and Lung Dysfunction during Klebsiella pneumoniae Infection in Mice

Klebsiella pneumoniae is an important pathogen associated with hospital-acquired pneumonia (HAP). Bacterial pneumonia is characterized by a harmful inflammatory response with a massive influx of neutrophils, production of cytokines and chemokines, and consequent tissue damage and dysfunction. Targeted therapies to block neutrophil migration to avoid tissue damage while keeping the antimicrobial properties of tissue remains a challenge in the field. Here we tested the effect of the anti-inflammatory properties of the chemokine fragment CXCL9(74–103) in pneumonia induced by Klebsiella pneumoniae in mice. Mice were infected by intratracheal injection of Klebsiella pneumoniae and 6 h after infection were treated systemically with CXCL9(74–103). The recruitment of leukocytes, levels of cytokines and chemokines, colony-forming units (CFU), and lung function were evaluated. The treatment with CXCL9(74–103) decreased neutrophil migration to the airways and the production of the cytokine interleukin-1β (IL-1β) without affecting bacterial control. In addition, the therapeutic treatment improved lung function in infected mice. Our results indicated that the treatment with CXCL9(74–103) reduced inflammation and improved lung function in Klebsiella pneumoniae-induced pneumonia.     

维生素C和E对Klebsiella pneumoniae合成1,3-丙二醇的调控

通过外源添加还原剂的方式调控细胞内NADH/NAD再生系统的状态,研究了40~150 mg/L VC及20~100 mg/L VE对Klebsiella penumoniae合成1,3-丙二醇的影响,发现外源添加150 mg/L VC或30 mg/L VE均可使1,3-PD合成浓度提高20%~30%;但同时也提高了某些副产物的合成浓度,对代谢流分布的调控作用不明显;1,3-丙二醇得率稍有提高但不显著. 提高1,3-PD得率宜从代谢节点(丙酮酸)通量调节方面考虑.     

Over‐expression of glycerol dehydrogenase and 1,3‐propanediol oxidoreductase in Klebsiella pneumoniae and their effects on conversion of glycerol into 1,3‐propanediol in resting cell system

BACKGROUND: Glycerol dehydrogenase [EC.1.1.1.6] and 1,3‐propanediol oxidoreductase [EC.1.1.1.202] were proved to be two of the key enzymes for glycerol conversion to 1,3‐propanediol in Klebsiella pneumoniae under anaerobic conditions. For insight into their significance on 1,3‐propanediol production under micro‐aerobic conditions, these two enzymes were over‐expressed in K. pneumoniae individually, and their effects on conversion of glycerol into 1,3‐propanediol in a resting cell system under micro‐aerobic conditions were investigated. RESULTS: In the resting cell system, over‐expression of 1,3‐propanediol oxidoreductase led to faster glycerol conversion and 1,3‐propanediol production. After a 12 h conversion process, it improved the yield of 1,3‐propanediol by 20.4% (222.1 mmol L⁻¹ versus 184.4 mmol L⁻¹) and enhanced the conversion ratio of glycerol into 1,3‐propanediol from 50.8% to 59.8% (mol mol⁻¹). Over‐expression of glycerol dehydrogenase in K. pneumoniae had no significant influence both on 1,3‐propanediol yield and on the conversion ratio of glycerol into 1,3‐propanediol in the resting cell system. CONCLUSION: The results were important for an understanding of the significance of glycerol dehydrogenase and 1,3‐propanediol oxidoreductase in 1,3‐proanediol production under micro‐aerobic conditions, and for developing better strategies to improve 1,3‐propanediol yield. Copyright © 2008 Society of Chemical Industry     

Effect of Divalent Metal Ion on the Structure,Stability and Function of Klebsiella pneumoniae Nicotinate-Nucleotide Adenylyltransferase: Empirical and Computational Studies

The continuous threat of drug-resistant Klebsiella pneumoniae justifies identifying novel targets and developing effective antibacterial agents. A potential target is nicotinate nucleotide adenylyltransferase (NNAT), an indispensable enzyme in the biosynthesis of the cell-dependent metabolite, NAD⁺. NNAT catalyses the adenylation of nicotinamide/nicotinate mononucleotide (NMN/NaMN), using ATP to form nicotinamide/nicotinate adenine dinucleotide (NAD⁺/NaAD). In addition, it employs divalent cations for co-substrate binding and catalysis and has a preference for different divalent cations. Here, the biophysical structure of NNAT from K. pneumoniae (KpNNAT) and the impact of divalent cations on its activity, conformational stability and substrate-binding are described using experimental and computational approaches. The experimental study was executed using an enzyme-coupled assay, far-UV circular dichroism, extrinsic fluorescence spectroscopy, and thermal shift assays, alongside homology modelling, molecular docking, and molecular dynamic simulation. The structure of KpNNAT revealed a predominately α-helical secondary structure content and a binding site that is partially hydrophobic. Its substrates ATP and NMN share the same binding pocket with similar affinity and exhibit an energetically favourable binding. KpNNAT showed maximum activity and minimal conformational changes with Mg²⁺ as a cofactor compared to Zn²⁺, Cu²⁺ and Ni²⁺. Overall, ATP binding affects KpNNAT dynamics, and the dynamics of ATP binding depend on the presence and type of divalent cation. The data obtained from this study would serve as a basis for further evaluation towards designing structure-based inhibitors with therapeutic potential.