scholarly journals Phage Selective Pressure Reduces Virulence of Hypervirulent Klebsiella pneumoniae Through Mutation of the wzc Gene

2021 ◽  
Vol 12 ◽  
Author(s):  
Lingjie Song ◽  
Xianggui Yang ◽  
Jinwei Huang ◽  
Xiaokui Zhu ◽  
Guohui Han ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Genomic Analysis ◽  
Resistant Bacteria ◽  
Comparative Genomic ◽  
Wild Type ◽  
Bacteriophage Therapy ◽  
Deletion Mutations ◽  
Invasive Infections

Hypervirulent Klebsiella pneumoniae (hvKp), one of the major community-acquired pathogens, can cause invasive infections such as liver abscess. In recent years, bacteriophages have been used in the treatment of K. pneumoniae, but the characteristics of the phage-resistant bacteria produced in the process of phage therapy need to be evaluated. In this study, two Podoviridae phages, hvKpP1 and hvKpP2, were isolated and characterized. In vitro and in vivo experiments demonstrated that the virulence of the resistant bacteria was significantly reduced compared with that of the wild type. Comparative genomic analysis of monoclonal sequencing showed that nucleotide deletion mutations of wzc and wcaJ genes led to phage resistance, and the electron microscopy and mucoviscosity results showed that mutations led to the loss of the capsule. Meanwhile, animal assay indicated that loss of capsule reduced the virulence of hvKp. These findings contribute to a better understanding of bacteriophage therapy, which not only can kill bacteria directly but also can reduce the virulence of bacteria by phage screening.

scholarly journals Wzc and WcaJ mutations in hypervirulent Klebsiella pneumoniae lead to phage resistance at the cost of reduced virulence

2021 ◽  
Author(s):  
Lingjie Song ◽  
Xianggui Yang ◽  
Jinwei Huang ◽  
Xiaokui Zhu ◽  
Guohui Han ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Phage Therapy ◽  
Genomic Analysis ◽  
Resistant Bacteria ◽  
Phage Resistance ◽  
Comparative Genomic ◽  
Sequencing Analysis ◽  
Bacteriophage Therapy ◽  
Invasive Infections ◽  
The Cost

AbstractHypervirulent Klebsiella pneumoniae (hvKp) is one of the major community-acquired pathogens, which can cause invasive infections such as liver abscess. In recent years, bacteriophages have been used in the treatment of Klebsiella pneumoniae, but the characteristics of the phage resistant bacteria produced in the process of phage therapy need to be evaluated. In this study, two podoviridae phages, hvKpP1 and hvKpP2, were isolated and characterized. In vitro and in vivo experiments demonstrated that the virulence of the resistant bacteria was significantly reduced compared with that of the wild type. Comparative genomic analysis of monoclonal sequencing showed that nucleotide deletion mutations of wzc and wcaJ genes led to phage resistance, and the electron microscopy and mucoviscosity results showed that mutations led to the loss of the capsule, meanwhile, animal assay indicated that loss of capsule reduced the virulence of hvKp. The findings can contribute to a better understanding of that bacteriophage therapy can not only kill bacteria directly, but also reduce the virulence of bacteria by phage screening.ImportanceBacteriophages are considered potential therapeutic alternative to antibiotics; however host-evolved phage resistance has accounted for the resurgences of pathogens, meaning further measures are need to improve the efficacy of phage therapy. This study showed two phages capable of infecting hypervirulent K. pneumoniae and identified phage-resistant mutants whose virulence was significantly reduced. Gene sequencing analysis revealed that mutations of wzc and wcaJ gene, related to capsule synthesis, recovered phage resistance but reduced the virulence of hypervirulent K. pneumoniae.

scholarly journals A Frameshift Mutation in wcaJ Associated with Phage Resistance in Klebsiella pneumoniae

2020 ◽  
Vol 8 (3) ◽  
pp. 378 ◽  
Author(s):  
Demeng Tan ◽  
Yiyuan Zhang ◽  
Jinhong Qin ◽  
Shuai Le ◽  
Jingmin Gu ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Phage Therapy ◽  
Rapid Development ◽  
Phage Resistance ◽  
Comparative Genomic ◽  
Lytic Phage ◽  
Wild Type ◽  
Coding Region

Phage therapy is a potential and promising avenue for controlling the emergence and spread of multidrug-resistant (MDR) Klebsiella pneumoniae, however, the rapid development of anti-phage resistance has been identified as an obstacle to the development of phage therapy. Little is known about the mechanism employed by MDR K. pneumoniae strains and how they protect themselves from lytic phage predation in vitro and in vivo. In this study, comparative genomic analysis shows undecaprenyl-phosphate glucose-1-phosphate transferase (WcaJ), the initial enzyme catalyzing the biosynthesis of colanic acid, is necessary for the adsorption of phage 117 (Podoviridae) to the host strain Kp36 to complete its lytic life cycle. In-frame deletion of wcaJ alone was sufficient to provide phage 117 resistance in the Kp36 wild-type strain. Complementation assays demonstrated the susceptibility of phage 117, and the mucoid phenotype could be restored in the resistant strain Kp36-117R by expressing the wild-type version of wcaJ. Remarkably, we found that bacterial mobile genetic elements (insA and insB) block phage 117 infections by disrupting the coding region of wcaJ, thus preventing phage adsorption to its phage receptor. Further, we revealed that the wcaJ mutation likely occurred spontaneously rather than adapted by phage 117 predation under unfavorable environments. Taken together, our results address a crucial evolutionary question around the mechanisms of phage–host interactions, increasing our current understandings of anti-phage defense mechanisms in this important MDR pathogen.

scholarly journals Identification of a Candidate Streptococcus pneumoniae Core Genome and Regions of Diversity Correlated with Invasive Pneumococcal Disease

2006 ◽  
Vol 74 (8) ◽  
pp. 4766-4777 ◽  
Author(s):  
Caroline Obert ◽  
Jack Sublett ◽  
Deepak Kaushal ◽  
Ernesto Hinojosa ◽  
Theresa Barton ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Invasive Pneumococcal Disease ◽  
Pneumococcal Disease ◽  
Core Genome ◽  
Genomic Analysis ◽  
Community Acquired Pneumonia ◽  
Comparative Genomic ◽  
Virulence Determinants ◽  
Wild Type

ABSTRACT Streptococcus pneumoniae is a leading cause of community-acquired pneumonia and gram-positive sepsis. While multiple virulence determinants have been identified, the combination of features that determines the propensity of an isolate to cause invasive pneumococcal disease (IPD) remains unknown. In this study, we determined the genetic composition of 42 invasive and 30 noninvasive clinical isolates of serotypes 6A, 6B, and 14 by comparative genomic hybridization. Comparison of the present/absent gene matrix (i.e., comparative genomic analysis [CGA]) identified a candidate core genome consisting of 1,553 genes (73% of the TIGR4 genome), 154 genes whose presence correlated with the ability to cause IPD, and 176 genes whose presence correlated with the noninvasive phenotype. Genes identified by CGA were cross-referenced with the published signature-tagged mutagenesis studies, which served to identify core and IPD-correlated genes required for in vivo passage. Among these, two pathogenicity islands, region of diversity 8a (RD8a), which encodes a neuraminidase and V-type sodium synthase, and RD10, which encodes PsrP, a protein homologous to the platelet adhesin GspB in Streptococcus gordonii, were identified. Mice infected with a PsrP mutant were delayed in the development of bacteremia and demonstrated reduced mortality versus wild-type-infected controls. Finally, the presence of seven RDs was determined to correlate with the noninvasive phenotype, a finding that suggests some RDs may contribute to asymptomatic colonization. In conclusion, RDs are unequally distributed between invasive and noninvasive isolates, RD8a and RD10 are correlated with the propensity of an isolate to cause IPD, and PsrP is required for full virulence in mice.

scholarly journals Inhaled bacteriophage therapy in a porcine model of ventilator-associated pneumonia caused by pseudomonas aeruginosa.

2021 ◽  
Author(s):  
Antoine Guillon ◽  
Jeoffrey Pardessus ◽  
Guillaume L’Hostis ◽  
Cindy Fevre ◽  
Celine Barc ◽  
...  
Keyword(s):  
Mechanical Ventilation ◽  
Pseudomonas Aeruginosa ◽  
Phage Therapy ◽  
Antimicrobial Efficacy ◽  
Resistant Bacteria ◽  
Ventilator Associated Pneumonia ◽  
Bacteriophage Therapy ◽  
Log Reduction

Background and Purpose. Pseudomonas aeruginosa is a main cause of ventilator-associated pneumonia (VAP) with drug-resistant bacteria. Bacteriophage therapy has experienced resurgence to compensate for the limited development of novel antibiotics. However, phage therapy is limited to a compassionate use so far, resulting from lack of adequate studies in relevant pharmacological models. We used a pig model of VAP caused by P. aeruginosa that recapitulates essential features of human disease to study the antimicrobial efficacy of nebulized-phage therapy. Experimental Approach. (i) Lysis kinetic assays were performed to evaluate in vitro phage antibacterial efficacy against P. aeruginosa and select relevant combinations of lytic phages. (ii) The efficacy of the phage combinations was investigated in vivo (murine model of P. aeruginosa lung infection). (iii) We determined the optimal conditions to ensure efficient phage delivery by aerosol during mechanical ventilation. (iv) Lung antimicrobial efficacy of inhaled-phage therapy was evaluated in pigs, which were anesthetized, mechanically ventilated and infected with P. aeruginosa. Key Results. By selecting an active phage cocktail and optimizing aerosol delivery conditions, we were able to deliver high phage concentrations in the lungs, which resulted in a rapid and marked reduction in P. aeruginosa density (1.5 Log reduction, p<0.001). No phage was detected in the sera and urines throughout the experiment. Conclusion and Implications. Our findings demonstrated: (i) the feasibility of delivering large amounts of active phages by nebulization during mechanical ventilation, (ii) rapid control of in situ infection by inhaled bacteriophage in an experimental model of VAP with high translational value.

scholarly journals In VivoEvolution to Colistin Resistance by PmrB Sensor Kinase Mutation in KPC-Producing Klebsiella pneumoniae Is Associated with Low-Dosage Colistin Treatment

2014 ◽  
Vol 58 (8) ◽  
pp. 4399-4403 ◽  
Author(s):  
Antonio Cannatelli ◽  
Vincenzo Di Pilato ◽  
Tommaso Giani ◽  
Fabio Arena ◽  
Simone Ambretti ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Genomic Analysis ◽  
Amino Acid Position ◽  
Comparative Genomic ◽  
Sensor Kinase ◽  
Wild Type ◽  
Colistin Resistance ◽  
Content Type ◽  
Before And After ◽  
Low Dosage

ABSTRACTColistin is a key drug for the treatment of infections caused by extensively drug-resistant strains ofEnterobacteriaceaeproducing carbapenemases. However, the emergence of colistin resistance is being increasingly reported, especially amongKlebsiella pneumoniaestrains producing KPC-type carbapenemases (KPC-KP). In this work, we investigated colistin-susceptible (KPB-1) and colistin-resistant (KPB-2) sequential isolates obtained from a patient with a KPC-KP infection before and after low-dosage colistin treatment, respectively. By using a next-generation sequencing approach and comparative genomic analysis of the two isolates, we detected in KPB-2 a nonsynonymous nucleotide substitution in the gene encoding the PmrB sensor kinase, resulting in a leucine-to-arginine substitution at amino acid position 82. Compared with KPB-1, KPB-2 exhibited upregulated transcription ofpmrAand ofpmrK, which is part of thepmrHFIJKLMoperon responsible for modification of the colistin lipopolysaccharide target. Complementation with wild-typepmrBin KPB-2 restored colistin susceptibility and reduced the transcription ofpmrAandpmrKto basal levels, while expression of PmrBL82Rin KPB-1 did not alter colistin susceptibility or upregulatepmrAandpmrKexpression, confirming the dominance of wild-type PmrB versus the PmrBL82Rmutant. The present results indicated that PmrB mutations mediating colistin resistance may be selected during low-dosage colistin treatment. The colistin-resistant phenotype of KPB-2 was stable for up to 50 generations in the absence of selective pressure and was not associated with a significant fitness cost in a competition experiment.

scholarly journals PredictingIn VivoEfficacy of Therapeutic Bacteriophages Used To Treat Pulmonary Infections

2013 ◽  
Vol 57 (12) ◽  
pp. 5961-5968 ◽  
Author(s):  
Marine Henry ◽  
Rob Lavigne ◽  
Laurent Debarbieux
Keyword(s):  
Mouse Lung ◽  
Resistant Bacteria ◽  
Infection Model ◽  
Bacteriophage Therapy ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Personalized Approach ◽  
First Time

ABSTRACTThe potential of bacteriophage therapy to treat infections caused by antibiotic-resistant bacteria has now been well established using various animal models. While numerous newly isolated bacteriophages have been claimed to be potential therapeutic candidates on the basis ofin vitroobservations, the parameters used to guide their choice among billions of available bacteriophages are still not clearly defined. We made use of a mouse lung infection model and a bioluminescent strain ofPseudomonas aeruginosato compare the activitiesin vitroandin vivoof a set of nine different bacteriophages (PAK_P1, PAK_P2, PAK_P3, PAK_P4, PAK_P5, CHA_P1, LBL3, LUZ19, and PhiKZ). For seven bacteriophages, a good correlation was found betweenin vitroandin vivoactivity. While the remaining two bacteriophages were activein vitro, they were not sufficiently activein vivounder similar conditions to rescue infected animals. Based on the bioluminescence recorded at 2 and 8 h postinfection, we also define for the first time a reliable index to predict treatment efficacy. Our results showed that the bacteriophages isolated directly on the targeted host were the most efficientin vivo, supporting a personalized approach favoring an optimal treatment.

scholarly journals Type X collagen gene regulation by Runx2 contributes directly to its hypertrophic chondrocyte–specific expression in vivo

2003 ◽  
Vol 162 (5) ◽  
pp. 833-842 ◽  
Author(s):  
Qiping Zheng ◽  
Guang Zhou ◽  
Roy Morello ◽  
Yuqing Chen ◽  
Xavier Garcia-Rojas ◽  
...  
Keyword(s):  
Binding Sites ◽  
Genomic Analysis ◽  
Comparative Genomic ◽  
Collagen Gene ◽  
Runt Domain ◽  
Specific Expression ◽  
Hypertrophic Chondrocyte ◽  
In Vitro Transfection

The α1(X) collagen gene (Col10a1) is the only known hypertrophic chondrocyte–specific molecular marker. Until recently, few transcriptional factors specifying its tissue-specific expression have been identified. We show here that a 4-kb murine Col10a1 promoter can drive β-galactosidase expression in lower hypertrophic chondrocytes in transgenic mice. Comparative genomic analysis revealed multiple Runx2 (Runt domain transcription factor) binding sites within the proximal human, mouse, and chick Col10a1 promoters. In vitro transfection studies and chromatin immunoprecipitation analysis using hypertrophic MCT cells showed that Runx2 contributes to the transactivation of this promoter via its conserved Runx2 binding sites. When the 4-kb Col10a1 promoter transgene was bred onto a Runx2+/− background, the reporter was expressed at lower levels. Moreover, decreased Col10a1 expression and altered chondrocyte hypertrophy was also observed in Runx2 heterozygote mice, whereas Col10a1 was barely detectable in Runx2-null mice. Together, these data suggest that Col10a1 is a direct transcriptional target of Runx2 during chondrogenesis.

scholarly journals N-methyl-bacillithiol, a new metabolite discovered in theChlorobiaceae, indicates that bacillithiol and derivatives are widely phylogenetically distributed

2017 ◽  
Author(s):  
Jennifer Hiras ◽  
Sunil V. Sharma ◽  
Vidhyavathi Raman ◽  
Ryan A. J. Tinson ◽  
Miriam Arbach ◽  
...  
Keyword(s):  
Redox Homeostasis ◽  
Genomic Analysis ◽  
Sulfur Oxidation ◽  
Comparative Genomic ◽  
Molecular Formula ◽  
Gram Positive Bacteria ◽  
Redox Responsive ◽  
Functional Consequences

AbstractLow-molecular weight (LMW) thiols are metabolites that mediate redox homeostasis and the detoxification of chemical stressors in cells. LMW thiols are also thought to play a central role in sulfur oxidation pathways in phototrophic bacteria, including theChlorobiaceae. Fluorescent thiol labeling of metabolite extracts coupled with HPLC showed thatChlorobaculum tepidumcontained a novel LMW thiol with a mass of 412 ± 1 Da corresponding to a molecular formula of C14H24N2O10S. These data suggested the new thiol is closely related to bacillithiol (BSH), the major LMW thiol from low G+C% Gram-positive bacteria. By comparing the as-isolated bimane adduct with chemically synthesized candidate structures, theCba. tepidumthiol structure was identified as N-methyl-bacillithiol (N-Me-BSH), methylated on the cysteine nitrogen, a rarely observed modification in metabolism. Orthologs of bacillithiol biosynthetic genes in theCba. tepidumgenome were required for the biosynthesis of N-Me-BSH. Furthermore, the CT1040 gene product was genetically identified as the BSH N-methyltransferase. N-Me-BSH was found in allChlorobiexamined as well asPolaribactersp. strain MED152, a member of theBacteroidetes.A comparative genomic analysis indicated that BSH/N-Me-BSH is synthesized not only by members of theChlorobi,Bacteroidetes,Deinococcus-Thermus, andFirmicutes, but also byAcidobacteria,Chlamydiae,Gemmatimonadetes, andProteobacteria.Significance StatementHere, N-Me-BSH is shown to be a redox-responsive LMW thiol cofactor inCba. tepidumand the gene,nmbA, encoding the BSH N-methyltransferase responsible for its synthesis is identified. The co-occurrence of orthologs to BSH biosynthesis genes and bacillithiol N-methyltransferase was confirmed to correctly predict LMW thiol biosynthesis in phylogenetically distant genomes. The analysis indicates that BSH/N-Me-BSH are likely the most widely distributed class of LMW thiols in biology. This finding sheds light on the evolution of LMW thiol metabolism, which is central to redox homeostasis, regulation and stress resistance in all cellular life. It also sheds light on a rare chemical modification. N-Me-BSH is the fourth instance of cysteinyl nitrogen methylation in metabolism. Identification of the BSH N-methyltransferase reported here will enable detailedin vivoandin vitrodissection of the functional consequences of this modification. As a standalone N-methyltransferase, NmbA may be useful as a component of constructed biosynthetic pathways for novel product (bio)synthesis.

scholarly journals Thioproline formation as a driver of formaldehyde toxicity in Escherichia coli

2020 ◽  
Author(s):  
Jenelle A. Patterson ◽  
Hai He ◽  
Jacob S. Folz ◽  
Qiang Li ◽  
Mark A. Wilson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Damage Control ◽  
Genomic Analysis ◽  
Relevant Information ◽  
Comparative Genomic ◽  
Control Mechanisms ◽  
One Carbon Metabolism ◽  
Formaldehyde Toxicity

ABSTRACTFormaldehyde (HCHO) is a reactive carbonyl compound that formylates and cross-links proteins, DNA, and small molecules. It is of specific concern as a toxic intermediate in the design of engineered pathways involving methanol oxidation or formate reduction. The high interest in engineering these pathways is not, however, matched by engineering-relevant information on precisely why HCHO is toxic or on what damage-control mechanisms cells deploy to manage HCHO toxicity. The only well-defined mechanism for managing HCHO toxicity is formaldehyde dehydrogenase-mediated oxidation to formate, which is counterproductive if HCHO is a desired pathway intermediate. We therefore sought alternative HCHO damage-control mechanisms via comparative genomic analysis. This analysis associated homologs of the Escherichia coli pepP gene with HCHO-related one-carbon metabolism. Furthermore, deleting pepP increased the sensitivity of E. coli to supplied HCHO but not other carbonyl compounds. PepP is a proline aminopeptidase that cleaves peptides of the general formula X-Pro-Y, yielding X + Pro-Y. HCHO is known to react spontaneously with cysteine to form the close proline analog thioproline (thiazolidine-4-carboxylate), which is incorporated into proteins and hence into proteolytic peptides. We therefore hypothesized that thioproline-containing peptides are toxic and that PepP cleaves these aberrant peptides. Supporting this hypothesis, PepP cleaved the model peptide Ala-thioproline-Ala as efficiently as Ala-Pro-Ala in vitro and in vivo, and deleting pepP increased sensitivity to supplied thioproline. Our data thus (i) provide biochemical genetic evidence that thioproline formation contributes substantially to HCHO toxicity and (ii) make PepP a candidate damage-control enzyme for engineered pathways having HCHO as an intermediate.

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