Conformational ensembles in Klebsiella pneumoniae FimH impact uropathogenesis.

Klebsiella pneumoniae is an important pathogen causing difficult-to-treat urinary tract infections (UTIs). Over 1.5 million women per year suffer from recurrent UTI, reducing quality of life and causing substantial morbidity and mortality, especially in the hospital setting. Uropathogenic E. coli (UPEC) is the most prevalent cause of UTI. Like UPEC, K. pneumoniae relies on type 1 pili, tipped with the mannose-binding adhesin FimH, to cause cystitis. However, K. pneumoniae FimH is a poor binder of mannose, despite a mannose-binding pocket identical to UPEC FimH. FimH is composed of two domains that are in an equilibrium between tense (low-affinity) and relaxed (high-affinity) conformations. Substantial interdomain interactions in the tense conformation yield a low-affinity, deformed mannose-binding pocket, while domain-domain interactions are broken in the relaxed state, resulting in a high-affinity binding pocket. Using crystallography, we identified the structural basis by which domain-domain interactions direct the conformational equilibrium of K. pneumoniae FimH, which is strongly shifted toward the low-affinity tense state. Removal of the pilin domain restores mannose binding to the lectin domain, thus showing that poor mannose binding by K. pneumoniae FimH is not an inherent feature of the mannose-binding pocket. Phylogenetic analyses of K. pneumoniae genomes found that FimH sequences are highly conserved. However, we surveyed a collection of K. pneumoniae isolates from patients with long-term indwelling catheters and identified isolates that possessed relaxed higher-binding FimH variants, which increased K. pneumoniae fitness in bladder infection models, suggesting that long-term residence within the urinary tract may select for higher-binding FimH variants.

Mutations in the efflux regulator gene oqxR provide a simple genetic switch for antimicrobial resistance in Klebsiella pneumoniae.

Klebsiella pneumoniae is a pathogen of major concern in the global rise of antimicrobial resistance and has been implicated as a reservoir for the transfer of resistance genes between species. The upregulation of efflux pumps is a particularly concerning mechanism of resistance acquisition as, in many instances, a single point mutation can simultaneously provide resistance to a range of antimicrobials and biocides. The current study investigated mutations in oqxR, which encodes a negative regulator of the RND-family efflux pump genes, oqxAB, natively found in the chromosome of K. pneumoniae. Resistant mutants in four K. pneumoniae strains (KP6870155, NTUH-K2044, SGH10, and ATCC43816) were selected from single exposures to 30 µg/mL chloramphenicol and 12 mutants were selected for whole genome sequencing to identify mutations associated with resistance. Resistant mutants generated by single exposures to chloramphenicol, tetracycline, or ciprofloxacin at ≥4 X MIC were replica plated onto all three antibiotics to observe simultaneous cross-resistance to all compounds, indicative of a multidrug resistance phenotype. A variety of novel mutations, including single point mutations, deletions, and insertions, were found to disrupt oqxR leading to significant and simultaneous increases in resistance to chloramphenicol, tetracycline, and ciprofloxacin. The oqxAB-oqxR locus has been mobilized and dispersed on plasmids in many Enterobacteriaceae species and the diversity of these loci was examined to evaluate the evolutionary pressures acting on these genes. Comparison of the promoter regions of oqxR in plasmid-borne copies of the oqxR-oqxAB operon indicated that some constructs may produce truncated versions of the oqxR transcript, which may impact on oqxAB regulation and expression. In some instances, co-carriage of chromosomal and plasmid encoded oqxAB-oqxR was found in K. pneumoniae, implying that there is selective pressure to maintain and expand the efflux pump. Given that OqxR is a repressor of oqxAB, any mutation affecting its expression or function can lead to multidrug resistance. This is in contrast to antibiotic target site mutations that must occur in limited sequence space to be effective and not impact the fitness of the cell. Therefore, oqxR may act as a simple genetic switch to facilitate resistance via OqxAB mediated efflux.

Molecular Mechanism of Ciprofloxacin Translocation Through the Major Diffusion Channels of the ESKAPE Pathogens Klebsiella pneumoniae and Enterobacter cloacae.

Experimental studies on the translocation and accumulation of antibiotics in Gram-negative bacteria have revealed details of the properties that allow efficient permeation through bacterial outer membrane porins. Among the major outer membrane diffusion channels, OmpF has been extensively studied to understand the antibiotic translocation process. In a few cases, this knowledge has also helped to improve the efficacy of existing antibacterial molecules. However, the extension of these strategies to enhance the efficacy of other existing and novel drugs require comprehensive molecular insight into the permeation process and an understanding of how antibiotic and channel properties influence the effective permeation rates. Previous studies have investigated how differences in antibiotic charge distribution can influence the observed permeation pathways through the OmpF channel, and have shown that the dynamics of the L3 loop can play a dominant role in the permeation process. Here, we perform all-atom simulations of the OmpF orthologs, OmpE35 from Enterobacter cloacae and OmpK35 from Klebsiella pneumoniae. Unbiased simulations of the porins and biased simulations of the ciprofloxacin permeation processes through these channels provide insight into the differences in the permeation pathway and energetics. In addition, we show that similar to the OmpF channel, antibiotic-induced dynamics of the L3 loop are also operative in the orthologs. However, the sequence and structural differences, influence the extent of the L3 loop fluctuations with OmpK35 showing greater stability in unbiased runs and subdued fluctuations in simulations with ciprofloxacin.

[Metabolic engineering of Klebsiella pneumoniae for 1, 3-propanediol production].

1, 3-propanediol is an important monomer for the production of polytrimethylene terephthalate (PTT). Currently, it is mainly produced by microbial fermentation, which, however, has low production efficiency. To address this problem, this study employed atmospheric room temperature plasma (ARTP) mutagenesis technology and high-throughput screening to obtain a strain with high tolerance to osmotic pressure, which achieved a 1, 3-propanediol titer of 87 g/L. Furthermore, the gene expression elements suitable for Klebsiella pneumoniae were screened, and metabolic engineering was employed to block redundant metabolic pathways (deletion of ldhA, budA, and aldA) and enhance the synthesis pathway (overexpression of dhaB and yqhD). The titer of 1, 3-propanediol produced by the engineered strain increased to 107 g/L. Finally, in a 5 L fermenter, the optimal strain KP-FMME-6 achieved a 1, 3-propanediol titer of 118 g/L, with a glycerol conversion rate of 42% and productivity of 2.46 g/(h·L), after optimization of the fermentation parameters. This study provides a reference for the industrial production of 1, 3-propanediol.

Insights into the recognition of hypermucoviscous Klebsiella pneumoniae clinical isolates by innate immune lectins of the Siglec and galectin families.

Klebsiella pneumoniae is an opportunistic bacterium that frequently colonizes the nasopharynx and gastrointestinal tract and can also cause severe infections when invading other tissues, particularly in immunocompromised individuals. Moreover, K. pneumoniae variants exhibiting a hypermucoviscous (HMV) phenotype are usually associated with hypervirulent strains that can produce invasive infections even in immunocompetent individuals. Major carbohydrate structures displayed on the K. pneumoniae surface are the polysaccharide capsule and the lipopolysaccharide, which presents an O-polysaccharide chain in its outermost part. Various capsular and O-chain structures have been described. Of note, production of a thick capsule is frequently observed in HMV variants. Here we examined the surface sugar epitopes of a collection of HMV and non-HMV K. pneumoniae clinical isolates and their recognition by several Siglecs and galectins, two lectin families of the innate immune system, using bacteria microarrays as main tool. No significant differences among isolates in sialic acid content or recognition by Siglecs were observed. In contrast, analysis of the binding of model lectins with diverse carbohydrate-binding specificities revealed striking differences in the recognition by galactose- and mannose-specific lectins, which correlated with the binding or lack of binding of galectins and pointed to the O-chain as the plausible ligand. Fluorescence microscopy and microarray analyses of galectin-9 binding to entire cells and outer membranes of two representative HMV isolates supported the bacteria microarray results. In addition, Western blot analysis of the binding of galectin-9 to outer membranes unveiled protein bands recognized by this galectin, and fingerprint analysis of these bands identified several proteins containing potential O-glycosylation sites, thus broadening the spectrum of possible galectin ligands on the K. pneumoniae surface. Moreover, Siglecs and galectins apparently target different structures on K. pneumoniae surfaces, thereby behaving as non-redundant complementary tools of the innate immune system.

RNA interactome of hypervirulent Klebsiella pneumoniae reveals a small RNA inhibitor of capsular mucoviscosity and virulence.

Hypervirulent Klebsiella pneumoniae (HvKP) is an emerging bacterial pathogen causing invasive infection in immune-competent humans. The hypervirulence is strongly linked to the overproduction of hypermucoviscous capsule, but the underlying regulatory mechanisms of hypermucoviscosity (HMV) have been elusive, especially at the post-transcriptional level mediated by small noncoding RNAs (sRNAs). Using a recently developed RNA interactome profiling approach iRIL-seq, we interrogate the Hfq-associated sRNA regulatory network and establish an intracellular RNA-RNA interactome in HvKP. Our data reveal numerous interactions between sRNAs and HMV-related mRNAs, and identify a plethora of sRNAs that repress or promote HMV. One of the strongest HMV repressors is ArcZ, which is activated by the catabolite regulator CRP and targets many HMV-related genes including mlaA and fbp. We discover that MlaA and its function in phospholipid transport is crucial for capsule retention and HMV, inactivation of which abolishes Klebsiella virulence in mice. ArcZ overexpression drastically reduces bacterial burden in mice and reduces HMV in multiple hypervirulent and carbapenem-resistant clinical isolates, indicating ArcZ is a potent RNA inhibitor of bacterial pneumonia with therapeutic potential. Our work unravels a novel CRP-ArcZ-MlaA regulatory circuit of HMV and provides mechanistic insights into the posttranscriptional virulence control in a superbug of global concern.

Microaerobic-mediated suppression of Klebsiella pneumoniae mucoviscosity is restored by rmpD overexpression.

AIMS: Hypervirulent Klebsiella pneumoniae (hvKp) causes invasive community-acquired infections in healthy individuals, and hypermucoviscosity (HMV) is the main phenotype associated with hvKp. This study investigates the impact of microaerobic environment availability on the mucoviscosity of K. pneumoniae. METHODS AND RESULTS: By culturing 25 clinical strains under microaerobic and aerobic environments, we observed a notable reduction in mucoviscosity in microaerobic environments. RNA sequencing and qRT-PCR revealed downregulated expressions of capsule synthesis genes (galf, orf2, wzi, wza, wzb, wzc, wcaj, manC, manB, and ugd) and regulatory genes (rmpA, rmpD, and rmpC) under microaerobic conditions. Transmission electron microscopy and Indian ink staining analysis were performed, revealing that the capsular thickness of K. pneumoniae decreased by half in microaerobic conditions compared to aerobic conditions. Deletion of rmpD and rmpC caused the loss of the HMV phenotype in both aerobic and microaerobic conditions. However, compared to wild-type strain in microaerobic condition, only rmpD overexpression strain, and not rmpC overexpression strain, displayed a significant increase in capsule thickness in microaerobic conditions. CONCLUSIONS: Microaerobic conditions can suppress the mucoviscosity of K. pneumoniae, but this suppression can be overcome by altering the expression of rmpD, indicating a specific function for rmpD in the oxygen environmental adaptation of K. pneumoniae.

Downregulation of Klebsiella pneumoniae RND efflux pump genes following indole signal produced by Escherichia coli.

BACKGROUND: More than a century has passed since it was discovered that many bacteria produce indole, but research into the actual biological roles of this molecule is just now beginning. The influence of indole on bacterial virulence was extensively investigated in indole-producing bacteria like Escherichia coli. To gain a deeper comprehension of its functional role, this study investigated how indole at concentrations of 0.5-1.0 mM found in the supernatant of Escherichia coli stationary phase culture was able to alter the virulence of non-indole-producing bacteria, such as Pseudomonas aeruginosa, Proteus mirabilis, and Klebsiella pneumoniae, which are naturally exposed to indole in mixed infections with Escherichia coli. RESULTS: Biofilm formation, antimicrobial susceptibility, and efflux pump activity were the three phenotypic tests that were assessed. Indole was found to influence antibiotic susceptibly of Pseudomonas aeruginosa, Proteus mirabilis and Klebsiella pneumoniae to ciprofloxacin, imipenem, ceftriaxone, ceftazidime, and amikacin through significant reduction in MIC with fold change ranged from 4 to 16. Biofilm production was partially abrogated in both 32/45 Pseudomonas aeruginosa and all eight Proteus mirabilis, while induced biofilm production was observed in 30/40 Klebsiella pneumoniae. Moreover, acrAB and oqxAB, which encode four genes responsible for resistance-nodulation-division multidrug efflux pumps in five isolates of Klebsiella pneumoniae were investigated genotypically using quantitative real-time (qRT)-PCR. This revealed that all four genes exhibited reduced expression indicated by 2^-ΔΔCT < 1 in indole-treated isolates compared to control group. CONCLUSION: The outcomes of qRT-PCR investigation of efflux pump expression have established a novel clear correlation of the molecular mechanism that lies beneath the influence of indole on bacterial antibiotic tolerance. This research provides novel perspectives on the various mechanisms and diverse biological functions of indole signaling and how it impacts the pathogenicity of non-indole-producing bacteria.

Evaluation of synergism effect of human glucose-dependent insulinotropic polypeptide (GIP) on Klebsiella pneumoniae carbapenemases (KPC) producer isolated from clinical samples.

Antibiotic resistance is increasing among Gram-negative bacteria, prompting the development of new antibiotics as well as alternative treatment approaches. Klebsiella pneumoniae Carbapenemases (KPC) has become a major concern in the treatment of infections, since KPC-producing bacteria are resistant to a number of ß -lactam and non ß-lactam antibiotics in addition to hydrolyzing carbapenemases. The aim of this study is to examine the synergistic effect of human Glucose-dependent Insulinotropic Polypeptide (GIP) on KPC producer. The K. pneumoniae isolates were identified by using biochemical tests and PCR genotyping. The disc diffusion method was used to assess the antimicrobial susceptibility of each isolate, and the modified Hodge test (MHT) was used to find carbapenemases. Agar well diffusion and minimum inhibitory concentration (MIC) assays were used to validate the synergistic effect of GIP against Klebsiella species. MIC values of chosen antimicrobial compounds demonstrated a considerable synergism impact when combined with human GIP, particularly against KPC strains. The antibacterial activity of the antimicrobial compounds was boosted by 4-16 times due to human GIP, reducing the MIC values. The fractional inhibitory concentration (FIC) ranged from 0.032 to 0.25 for examined antibiotics. Thus, GIP can be considered an antibacterial adjuvant with the potential to supplement the current antibiotic spectrum.

The transcriptional regulator Fur modulates the expression of uge, a gene essential for the core lipopolysaccharide biosynthesis in Klebsiella pneumoniae.

BACKGROUND: Klebsiella pneumoniae is a Gram-negative pathogen that has become a threat to public health worldwide due to the emergence of hypervirulent and multidrug-resistant strains. Cell-surface components, such as polysaccharide capsules, fimbriae, and lipopolysaccharides (LPS), are among the major virulence factors for K. pneumoniae. One of the genes involved in LPS biosynthesis is the uge gene, which encodes the uridine diphosphate galacturonate 4-epimerase enzyme. Although essential for the LPS formation in K. pneumoniae, little is known about the mechanisms that regulate the expression of uge. Ferric uptake regulator (Fur) is an iron-responsive transcription factor that modulates the expression of capsular and fimbrial genes, but its role in LPS expression has not yet been identified. This work aimed to investigate the role of the Fur regulator in the expression of the K. pneumoniae uge gene and to determine whether the production of LPS by K. pneumoniae is modulated by the iron levels available to the bacterium. RESULTS: Using bioinformatic analyses, a Fur-binding site was identified on the promoter region of the uge gene; this binding site was validated experimentally through Fur Titration Assay (FURTA) and DNA Electrophoretic Mobility Shift Assay (EMSA) techniques. RT-qPCR analyses were used to evaluate the expression of uge according to the iron levels available to the bacterium. The iron-rich condition led to a down-regulation of uge, while the iron-restricted condition resulted in up-regulation. In addition, LPS was extracted and quantified on K. pneumoniae cells subjected to iron-replete and iron-limited conditions. The iron-limited condition increased the amount of LPS produced by K. pneumoniae. Finally, the expression levels of uge and the amount of the LPS were evaluated on a K. pneumoniae strain mutant for the fur gene. Compared to the wild-type, the strain with the fur gene knocked out presented a lower LPS amount and an unchanged expression of uge, regardless of the iron levels. CONCLUSIONS: Here, we show that iron deprivation led the K. pneumoniae cells to produce higher amount of LPS and that the Fur regulator modulates the expression of uge, a gene essential for LPS biosynthesis. Thus, our results indicate that iron availability modulates the LPS biosynthesis in K. pneumoniae through a Fur-dependent mechanism.

H-NS involved in positive regulation of glycerol dehydratase gene expression in Klebsiella pneumoniae 2e.

Glycerol dehydratase is the key and rate-limiting enzyme in the 1,3-propanediol synthesis pathway of Klebsiella pneumoniae, which determined the producing rate and yield of 1,3-propanediol. However, the expression regulation mechanism of glycerol dehydratase gene dhaB remains poorly unknown. In this study, a histone-like nucleoid-structuring (H-NS) protein was identified and characterized as the positive transcription regulator for dhaB expression in K. pneumoniae 2e, which exhibited high tolerance against crude glycerol in our previous study. Deletion of hns gene significantly decreased the transcription level of dhaB in K. pneumoniae 2e, which led to a remarkable defect on strain growth, glycerol dehydratase activity, and 3-hydroxypropanal production during glycerol fermentation. The transcription level of dhaB was significantly up-regulated in crude glycerol relative to pure glycerol, while the inactivation of H-NS resulted in more negative effect for transcription level of dhaB in the former. Though the H-NS expression level was almost comparable in both substrates, its multimer state was reduced in crude glycerol relative to pure glycerol, suggesting that the oligomerization state of H-NS might have contributed for positive regulation of dhaB expression. Furthermore, electrophoretic mobility shift and DNase I footprinting assays showed that H-NS could directly bind to the upstream promoter region of dhaB by recognizing the AT-rich region. These findings provided new insight into the transcriptional regulation mechanism of H-NS for glycerol dehydratase expression in K. pneumoniae, which might offer new target for engineering bacteria to industrially produce 1,3-propanediol.IMPORTANCEThe biological production of 1,3-propanediol from glycerol by microbial fermentation shows great promising prospect on industrial application. Glycerol dehydratase catalyzes the penultimate step in glycerol metabolism and is regarded as one of the key and rate-limiting enzymes for 1,3-propanediol production. H-NS was reported as a pleiotropic modulator with negative effects on gene expression in most studies. Here, we reported for the first time that the expression of glycerol dehydratase gene is positively regulated by the H-NS. The results provide insight into a novel molecular mechanism of H-NS for positive regulation of glycerol dehydratase gene expression in K. pneumoniae, which holds promising potential for facilitating construction of engineering highly efficient 1,3-propanediol-producing strains.

Small RNA-regulated expression of efflux pump affects tigecycline resistance and heteroresistance in clinical isolates of Klebsiella pneumoniae.

Tigecycline and the newly Food and Drug Administration-approved tetracyclines, including eravacycline and omadacycline, are regarded as last-resort treatments for multidrug-resistant Enterobacterales. However, tigecycline resistance in Klebsiella pneumoniae has increased, especially the underlying mechanism of heteroresistance is unclear. This study aimed to elucidate the mechanisms underlying tigecycline resistance and heteroresistance in clinical K. pneumoniae isolates. A total of 153 clinical K. pneumoniae isolates were collected, and identified 15 tigecycline-resistant and three tigecycline-heteroresistant isolates using broth microdilution and population analysis profile methods, respectively. Total RNAs from K. pneumoniae ATCC13883 and the laboratory-induced tigecycline-resistant strain were extracted and sequenced on an Illumina platform. Differentially expressed genes and regulatory small RNAs (sRNAs) were analyzed and validated in clinical isolates of K. pneumoniae using quantitative real-time PCR. RNA sequencing results showed that mdtABC efflux pump genes were significantly upregulated in the tigecycline-resistant strains. Overexpression of mdtABC was observed in a clinical K. pneumoniae isolate, which increased tigecycline minimum inhibitory concentrations (MICs) and was involved in tigecycline heteroresistance. Sequencing analysis of sRNA demonstrated that candidate sRNA-120 directly interacted with the mdtABC operon and was downregulated in tigecycline-resistant strains. We generated an sRNA-120 deletion mutation strain and a complemented strain of K. pneumoniae. The sRNA-120 deletion strain displayed increased mRNA levels of mdtA, mdtB, and mdtC and an increase in MICs of tigecycline. The complemented strain of sRNA-120 restored the mRNA levels of these genes and the susceptibility to tigecycline. RNA antisense purification and parallel reaction monitoring mass spectrometry were performed to verify the interactions between sRNA-120 and mdtABC. Collectively, our study highlights that the post-transcriptional repression of mdtABC through sRNA-120 may provide an additional layer of efflux pump gene expression control, which is important for resistance and heteroresistance in clinical K. pneumoniae isolates.

Gas Chromatography-Ion Mobility Spectrometry Reveals Acetoin as a Biomarker for Carbapenemase-Producing Klebsiella pneumoniae.

BACKGROUND This study aimed to detect the volatile organic compound (VOC), 3-hydroxy-2-butanone (acetoin) using gas chromatography-ion mobility spectrometry (GC-IMS) in antimicrobial-resistant Klebsiella pneumoniae (K. pneumoniae) carbapenemase (KPC)-producing bacteria. MATERIAL AND METHODS Using stromal fluid of blood culture bottles (BacT/ALERT® SA) as the medium, 3-hydroxy-2-butanone (acetoin) released by K. pneumoniae during growth was detected using GC-IMS. The impact of imipenem (IPM) and carbapenemase inhibitors [avibactam sodium or pyridine-2,6-dicarboxylic acid (DPA)] on the emission of 3-hydroxy-2-butanone (acetoin) from various carbapenemase-producing K. pneumoniae was further investigated. Subsequently, VOCal software was used to generate a pseudo-3D plot of 3-hydroxy-2-butanone (acetoin), and the relative peak volumes were exported for data analysis. Standard strains served as references, and the findings were validated with clinical isolates. RESULTS The pattern of temporal changes in the 3-hydroxy-2-butanone (acetoin) release from K. pneumoniae in the absence of IPM was consistent with the growth curve. After the IPM addition, carbapenemase-positive strains released significantly higher contents of 3-hydroxy-2-butanone (acetoin) than carbapenemase-negative strains at the late exponential growth phase (T2). Notably, adding avibactam sodium significantly decreased the 3-hydroxy-2-butanone (acetoin) content released from the class A carbapenemase-producing strains as compared to the absence of the carbapenemase inhibitor. Conversely, adding DPA significantly decreased the 3-hydroxy-2-butanone (acetoin) content released from the class B carbapenemase-producing strains (both standard and clinical strains, all P<0.05). CONCLUSIONS This study demonstrated the potential of 3-hydroxy-2-butanone (acetoin) as a VOC biomarker for detecting carbapenemase-producing K. pneumoniae, as revealed by GC-IMS analysis.

Expression, Purification and Biophysical Characterisation of Klebsiella Pneumoniae Protein Adenylyltransferase: A Systematic Integration of Empirical and Computational Modelling Approaches.

Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. Klebsiella pneumoniae (K. pneumoniae) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg2+ binding to Klebsiella pneumoniae PrAT (KpPrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying KpPrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg2+. Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg2+, while mant-ATP studies allude to the potential nucleotide binding ability of KpPrAT. The presence of Mg2+ increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to KpPrAT, with or without Mg2+. Conclusively, the presence of Mg2+ induces a conformation in KpPrAT that favours nucleotide binding.

High production of enantiopure (R,R)-2,3-butanediol from crude glycerol by Klebsiella pneumoniae with an engineered oxidative pathway and a two-stage agitation strategy.

BACKGROUND: (R,R)-2,3-butanediol (BDO) is employed in a variety of applications and is gaining prominence due to its unique physicochemical features. The use of glycerol as a carbon source for 2,3-BDO production in Klebsiella pneumoniae has been limited, since 1,3-propanediol (PDO) is generated during glycerol fermentation. RESULTS: In this study, the inactivation of the budC gene in K. pneumoniae increased the production rate of (R,R)-2,3-BDO from 21.92 ± 2.10 to 92.05 ± 1.20%. The major isomer form of K. pneumoniae (meso-2,3-BDO) was shifted to (R,R)-2,3-BDO. The purity of (R,R)-2,3-BDO was examined by agitation speed, and 98.54% of (R,R)-2,3-BDO was obtained at 500 rpm. However, as the cultivation period got longer, the purity of (R,R)-2,3-BDO declined. For this problem, a two-step agitation speed control strategy (adjusted from 500 to 400 rpm after 24 h) and over-expression of the dhaD gene involved in (R,R)-2,3-BDO biosynthesis were used. Nevertheless, the purity of (R,R)-2,3-BDO still gradually decreased over time. Finally, when pure glycerol was replaced with crude glycerol, the titer of 89.47 g/L of (R,R)-2,3-BDO (1.69 g/L of meso-2,3-BDO), productivity of 1.24 g/L/h, and yield of 0.35 g/g consumed crude glycerol was achieved while maintaining a purity of 98% or higher. CONCLUSIONS: This study is meaningful in that it demonstrated the highest production and productivity among studies in that produced (R,R)-2,3-BDO with a high purity in Klebsiella sp. strains. In addition, to the best of our knowledge, this is the first study to produce (R,R)-2,3-BDO using glycerol as the sole carbon source.

Biological characteristics of manganese transporter MntP in Klebsiella pneumoniae.

Klebsiella pneumoniae is an important opportunistic pathogen that causes a variety of infections. It is critical for bacteria to maintain metal homeostasis during infection. By using an isogenic mntP deletion mutant of K. pneumoniae strain NTUH-K2044, we found that MntP was a manganese efflux pump. Manganese increased the tolerance to oxidative stress, and oxidative stress could increase the intracellular manganese concentration. In oxidative stress, the mntP deletion mutant exhibited significantly higher sensitivity to manganese. Furthermore, iron could increase the tolerance of the mntP deletion mutant to manganese. Inductively coupled plasma mass spectrometry analysis revealed that the mntP deletion mutant had higher intracellular manganese and iron concentrations than wild-type and complementary strains. These findings suggested that iron could increase manganese tolerance in K. pneumoniae. This work elucidated the role of MntP in manganese detoxification and Mn/Fe homeostasis in K. pneumoniae.IMPORTANCEMetal homeostasis plays an important role during the process of bacterial infection. Herein, we revealed that MntP was involved in intracellular manganese homeostasis. Manganese promoted resistance to oxidative stress in Klebsiella pneumoniae. Furthermore, we demonstrated that the mntP deletion mutant exhibited significantly lower survival under manganese and H2O2 conditions. Oxidative stress increased the intracellular manganese content of the mntP deletion mutant. MntP played a critical role in maintaining intracellular manganese and iron concentrations. MntP contributed to manganese detoxification and Mn/Fe homeostasis in K. pneumoniae.

Protein function annotation and virulence factor identification of Klebsiella pneumoniae genome by multiple machine learning models.

Klebsiella pneumoniae is a type of Gram-negative bacterium which can cause a range of infections in human. In recent years, an increasing number of strains of K. pneumoniae resistant to multiple antibiotics have emerged, posing a significant threat to public health. The protein function of this bacterium is not well known, thus a systematic investigation of K. pneumoniae proteome is in urgent need. In this study, the protein functions of this bacteria were re-annotated, and their function groups were analyzed. Moreover, three machine learning models were built to identify novel virulence factors. Results showed that the functions of 16 uncharacterized proteins were first annotated by sequence alignment. In addition, K. pneumoniae proteins share a high proportion of homology with Haemophilus influenzae and a low homology proportion with Chlamydia pneumoniae. By sequence analysis, 10 proteins were identified as potential drug targets for this bacterium. Our model achieved a high accuracy of 0.901 in the benchmark dataset. By applying our models to K. pneumoniae, we identified 39 virulence factors in this pathogen. Our findings could provide novel clues for the treatment of K. pneumoniae infection.

AcrAB-TolC efflux pump overexpression and tet(A) gene mutation increase tigecycline resistance in Klebsiella pneumoniae.

Tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) is increasing and has emerged as a global public health issue. However, the mechanism of tigecycline resistance remains unclear. The objective of this study was to investigate the potential role of efflux pump system in tigecycline resistance. 29 tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) strains were collected and their minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. The ramR, acrR, rpsJ, tet(A), and tet(X) were amplified by polymerase chain reaction (PCR). The mRNA expression of different efflux pump genes and regulator genes were analyzed by real-time PCR. Additionally, KP14 was selected for genome sequencing. KP14 genes without acrB, oqxB, and TetA were modified using suicide plasmids and MIC of tigecycline of KP14 with target genes knocked out was investigated. It was found that MIC of tigecycline of 20 out of the 29 TNSKP strains decreased by over four folds once combined with phenyl-arginine-ß-naphthylamide dihydrochloride (PaßN). Most strains exhibited upregulation of AcrAB and oqxAB efflux pumps. The strains with acrB, oqxB, and tetA genes knocked out were constructed, wherein the MIC of tigecycline of KP14∆acrB and KP14∆tetA was observed to be 2 µg/mL (decreased by 16 folds), the MIC of tigecycline of KP14ΔacrBΔTetA was 0.25 µg/mL (decreased by 128 folds), but the MIC of tigecycline of KP14∆oqxB remained unchanged at 32 µg/mL. The majority of TNSKP strains demonstrated increased expression of AcrAB-TolC and oqxAB, while certain strains showed mutations in other genes associated with tigecycline resistance. In KP14, both overexpression of AcrAB-TolC and tet(A) gene mutation contributed to the mechanism of tigecycline resistance.

RIL-seq reveals extensive involvement of small RNAs in virulence and capsule regulation in hypervirulent Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae (hvKp) can infect healthy individuals, in contrast to classical strains that commonly cause nosocomial infections. The recent convergence of hypervirulence with carbapenem-resistance in K. pneumoniae can potentially create 'superbugs' that are challenging to treat. Understanding virulence regulation of hvKp is thus critical. Accumulating evidence suggest that posttranscriptional regulation by small RNAs (sRNAs) plays a role in bacterial virulence, but it has hardly been studied in K. pneumoniae. We applied RIL-seq to a prototypical clinical isolate of hvKp to unravel the Hfq-dependent RNA-RNA interaction (RRI) network. The RRI network is dominated by sRNAs, including predicted novel sRNAs, three of which we validated experimentally. We constructed a stringent subnetwork composed of RRIs that involve at least one hvKp virulence-associated gene and identified the capsule gene loci as a hub target where multiple sRNAs interact. We found that the sRNA OmrB suppressed both capsule production and hypermucoviscosity when overexpressed. Furthermore, OmrB base-pairs within kvrA coding region and partially suppresses translation of the capsule regulator KvrA. This agrees with current understanding of capsule as a major virulence and fitness factor. It emphasizes the intricate regulatory control of bacterial phenotypes by sRNAs, particularly of genes critical to bacterial physiology and virulence.

Klebsiella pneumoniae O-polysaccharide biosynthesis highlights the diverse organization of catalytic modules in ABC transporter-dependent glycan assembly.

Klebsiella pneumoniae provides influential prototypes for lipopolysaccharide O antigen (OPS) biosynthesis in Gram-negative bacteria. Sequences of OPS-biosynthesis gene clusters in serotypes O4 and O7 suggest fundamental differences in the organization of required enzyme modules compared to other serotypes. Furthermore, some required activities were not assigned by homology shared with characterized enzymes. The goal of this study was therefore to resolve the serotype O4 and O7 pathways to expand our broader understanding of glycan polymerization and chain termination processes. The O4 and O7 antigens were produced from cloned genetic loci in recombinant Escherichia coli. Systematic in vivo and in vitro approaches were then applied to assign each enzyme in each of the pathways, defining the necessary components for polymerization and chain termination. OPS assembly is accomplished by multiprotein complexes formed by interactions between polymerase components variably distributed in single and multimodule proteins. In each complex, a terminator function is present in a protein containing a characteristic coiled-coil molecular ruler, which determines glycan chain length. In serotype O4, we discovered a CMP-α-3-deoxy-ᴅ-manno-octulosonic acid-dependent chain-terminating glycosyltransferase that is the founding member of a new glycosyltransferase family (GT137) and potentially identifies a new glycosyltransferase fold. The O7 OPS is terminated by a methylphosphate moiety, like the K. pneumoniae O3 antigen, but the methyltransferase-kinase enzyme pairs responsible for termination in these serotypes differ in sequence and predicted structures. Together, the characterization of O4 and O7 has established unique enzyme activities and provided new insight into glycan-assembly strategies that are widely distributed in bacteria.