Identification of Two Regulators of Virulence That Are Conserved inKlebsiella pneumoniaeClassical and Hypervirulent Strains

ABSTRACTKlebsiella pneumoniaeis widely recognized as a pathogen with a propensity for acquiring antibiotic resistance. It is capable of causing a range of hospital-acquired infections (urinary tract infections [UTI], pneumonia, sepsis) and community-acquired invasive infections. The genetic heterogeneity ofK. pneumoniaeisolates complicates our ability to understand the virulence ofK. pneumoniae. Characterization of virulence factors conserved between strains as well as strain-specific factors will improve our understanding of this important pathogen. The MarR family of regulatory proteins is widely distributed in bacteria and regulates cellular processes such as antibiotic resistance and the expression of virulence factors.Klebsiellaencodes numerous MarR-like proteins, and they likely contribute to the ability ofK. pneumoniaeto respond to and survive under a wide variety of environmental conditions, including those present in the human body. We tested loss-of-function mutations in all themarRhomologues in a murine pneumonia model and found that two (kvrAandkvrB) significantly impacted the virulence of K1 and K2 capsule type hypervirulent (hv) strains and thatkvrAaffected the virulence of a sequence type 258 (ST258) classical strain. In thehvstrains,kvrAandkvrBmutants displayed phenotypes associated with reduced capsule production, mucoviscosity, and transcription fromgalFandmanCpromoters that drive expression of capsule synthesis genes. In contrast,kvrAandkvrBmutants in the ST258 strain had no effect on capsule gene expression or capsule-related phenotypes. Thus, KvrA and KvrB affect virulence in classical andhvstrains but the effect on virulence may not be exclusively due to effects on capsule production.IMPORTANCEIn addition to having a reputation as the causative agent for hospital-acquired infections as well as community-acquired invasive infections,Klebsiella pneumoniaehas gained widespread attention as a pathogen with a propensity for acquiring antibiotic resistance. Due to the rapid emergence of carbapenem resistance amongK. pneumoniaestrains, a better understanding of virulence mechanisms and identification of new potential drug targets are needed. This study identified two novel regulators (KvrA and KvrB) of virulence inK. pneumoniaeand demonstrated that their effect on virulence in invasive strains is likely due in part to effects on capsule production (a major virulence determinant) and hypermucoviscosity. KvrA also impacts the virulence of classical strains but does not appear to affect capsule gene expression in this strain. KvrA and KvrB are conserved amongK. pneumoniaestrains and thus could regulate capsule expression and virulence in diverse strains regardless of capsule type.

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A Serendipitous Mutation Reveals the Severe Virulence Defect of a Klebsiella pneumoniae fepB Mutant

mSphere ◽

10.1128/msphere.00341-17 ◽

2017 ◽

Vol 2 (4) ◽

Cited By ~ 12

Author(s):

Michelle Palacios ◽

Christopher A. Broberg ◽

Kimberly A. Walker ◽

Virginia L. Miller

Keyword(s):

Antibiotic Resistance ◽

Urinary Tract ◽

Klebsiella Pneumoniae ◽

Drug Targets ◽

Carbapenem Resistance ◽

Content Type ◽

Hospital Acquired Infections ◽

Tract Infections ◽

Hospital Acquired ◽

Rapid Emergence

ABSTRACT In addition to having a reputation as the causative agent of several types of hospital-acquired infections, Klebsiella pneumoniae has gained widespread attention as a pathogen with a propensity for acquiring antibiotic resistance. It is capable of causing a range of infections, including urinary tract infections, pneumonia, and sepsis. Because of the rapid emergence of carbapenem resistance among Klebsiella strains, there is a dire need for a better understanding of virulence mechanisms and identification of new drug targets. Here, we identify the periplasmic transporter FepB as one such potential target. Klebsiella pneumoniae is considered a significant public health threat because of the emergence of multidrug-resistant strains and the challenge associated with treating life-threatening infections. Capsule, siderophores, and adhesins have been implicated as virulence determinants of K. pneumoniae, yet we lack a clear understanding of how this pathogen causes disease. In a previous screen for virulence genes, we identified a potential new virulence locus and constructed a mutant (smr) with this locus deleted. In this study, we characterize the smr mutant and show that this mutation renders K. pneumoniae avirulent in a pneumonia model of infection. The smr mutant was expected to have a deletion of three genes, but subsequent genome sequencing indicated that a much larger deletion had occurred. Further analysis of the deleted region indicated that the virulence defect of the smr mutant could be attributed to the loss of FepB, a periplasmic protein required for import of the siderophore enterobactin. Interestingly, a ΔfepB mutant was more attenuated than a mutant unable to synthesize enterobactin, suggesting that additional processes are affected. As FepB is highly conserved among the members of the family Enterobacteriaceae, therapeutic targeting of FepB may be useful for the treatment of Klebsiella and other bacterial infections. IMPORTANCE In addition to having a reputation as the causative agent of several types of hospital-acquired infections, Klebsiella pneumoniae has gained widespread attention as a pathogen with a propensity for acquiring antibiotic resistance. It is capable of causing a range of infections, including urinary tract infections, pneumonia, and sepsis. Because of the rapid emergence of carbapenem resistance among Klebsiella strains, there is a dire need for a better understanding of virulence mechanisms and identification of new drug targets. Here, we identify the periplasmic transporter FepB as one such potential target.

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AKlebsiella pneumoniaeRegulatory Mutant Has Reduced Capsule Expression but Retains Hypermucoviscosity

mBio ◽

10.1128/mbio.00089-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 23

Author(s):

Kimberly A. Walker ◽

Taryn A. Miner ◽

Michelle Palacios ◽

Dominika Trzilova ◽

Daniel R. Frederick ◽

...

Keyword(s):

Gene Expression ◽

Health Care ◽

Klebsiella Pneumoniae ◽

Virulence Factors ◽

Virulence Factor ◽

New Drugs ◽

Content Type ◽

Novel Therapeutics ◽

Regulatory Cascade ◽

Capsule Production

ABSTRACTThe polysaccharide capsule is an essential virulence factor forKlebsiella pneumoniaein both community-acquired hypervirulent strains as well as health care-associated classical strains that are posing significant challenges due to multidrug resistance. Capsule production is known to be transcriptionally regulated by a number of proteins, but very little is known about how these proteins collectively control capsule production. RmpA and RcsB are two known regulators of capsule gene expression, and RmpA is required for the hypermucoviscous (HMV) phenotype in hypervirulentK. pneumoniaestrains. In this report, we confirmed that these regulators performed their anticipated functions in the ATCC 43816 derivative, KPPR1S:rcsBandrmpAmutants are HMV negative and have reduced capsule gene expression. We also identified a novel transcriptional regulator, RmpC, encoded by a gene nearrmpA. The ΔrmpCstrain has reduced capsule gene expression but retains the HMV phenotype. We further showed that a regulatory cascade exists in which KvrA and KvrB, the recently characterized MarR-like regulators, and RcsB contribute to capsule regulation through regulation of thermpApromoter and through additional mechanisms. In a murine pneumonia model, the regulator mutants have a range of colonization defects, suggesting that they regulate virulence factors in addition to capsule. Further testing of thermpCandrmpAmutants revealed that they have distinct and overlapping functions and provide evidence that HMV is not dependent on overproduction of capsule. This distinction will facilitate a better understanding of HMV and how it contributes to enhanced virulence of hypervirulent strains.IMPORTANCEKlebsiella pneumoniaecontinues to be a substantial public health threat due to its ability to cause health care-associated and community-acquired infections combined with its ability to acquire antibiotic resistance. Novel therapeutics are needed to combat this pathogen, and a greater understanding of its virulence factors is required for the development of new drugs. A key virulence factor forK. pneumoniaeis the capsule, and community-acquired hypervirulent strains produce a capsule that causes hypermucoidy. We report here a novel capsule regulator, RmpC, and provide evidence that capsule production and the hypermucoviscosity phenotype are distinct processes. Infection studies showing that this and other capsule regulator mutants have a range of phenotypes indicate that additional virulence factors are in their regulons. These results shed new light on the mechanisms controlling capsule production and introduce targets that may prove useful for the development of novel therapeutics for the treatment of this increasingly problematic pathogen.

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Novel Drivers of Virulence in Clostridioides difficile Identified via Context-Specific Metabolic Network Analysis

mSystems ◽

10.1128/msystems.00919-21 ◽

2021 ◽

Author(s):

Matthew L. Jenior ◽

Jhansi L. Leslie ◽

Deborah A. Powers ◽

Elizabeth M. Garrett ◽

Kimberly A. Walker ◽

...

Keyword(s):

Network Analysis ◽

Metabolic Network ◽

Virulence Factors ◽

Metabolic Pathways ◽

Content Type ◽

Hospital Acquired Infections ◽

Metabolic Network Analysis ◽

Clostridioides Difficile ◽

Context Specific ◽

Hospital Acquired

Clostridioides difficile has become the leading single cause of hospital-acquired infections. Numerous studies have demonstrated the importance of specific metabolic pathways in aspects of C. difficile pathophysiology, from initial colonization to regulation of virulence factors.

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Complete Genome Sequence of Klebsiella pneumoniae Phage Sweeny

Microbiology Resource Announcements ◽

10.1128/mra.01047-19 ◽

2019 ◽

Vol 8 (39) ◽

Cited By ~ 1

Author(s):

Nicholas Martinez ◽

Eric Williams ◽

Heather Newkirk ◽

Mei Liu ◽

Jason J. Gill ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Complete Genome Sequence ◽

Protein Level ◽

Multidrug Resistant ◽

Content Type ◽

Hospital Acquired Infections ◽

Multidrug Resistant Bacterium ◽

Protein Encoding ◽

Hospital Acquired ◽

Encoding Genes

Klebsiella pneumoniae is a multidrug-resistant bacterium causing many severe hospital-acquired infections. Here, we describe siphophage Sweeny that infects K. pneumoniae. Of its 78 predicted protein-encoding genes, a functional assignment was given to 36 of them. Sweeny is most closely related to T1-like phages at the protein level.

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Complete Genome Sequence of Klebsiella pneumoniae Myophage Mulock

Microbiology Resource Announcements ◽

10.1128/mra.01338-19 ◽

2019 ◽

Vol 8 (46) ◽

Author(s):

Lorna Min ◽

Lauren Lessor ◽

Chandler O’Leary ◽

Rachele Bonasera ◽

Jason Gill ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

Opportunistic Pathogen ◽

Phage Lambda ◽

Content Type ◽

Hospital Acquired Infections ◽

Genes Encoding ◽

Hospital Acquired

Klebsiella pneumoniae is an opportunistic pathogen associated with hospital-acquired infections. This report describes the complete genome of the K. pneumoniae myophage Mulock, which appears to be a temperate myophage distantly related to other Klebsiella myophages in morphogenesis genes and is partially syntenic with the canonical Escherichia phage lambda in genes encoding lambda-like functions.

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Complete Genome Sequence of a Multidrug-Resistant, blaNDM-1-Expressing Klebsiella pneumoniae K66-45 Clinical Isolate from Norway

Genome Announcements ◽

10.1128/genomea.00601-17 ◽

2017 ◽

Vol 5 (27) ◽

Cited By ~ 4

Author(s):

Adam Heikal ◽

Ørjan Samuelsen ◽

Tom Kristensen ◽

Ole Andreas Økstad

Keyword(s):

Klebsiella Pneumoniae ◽

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

Multidrug Resistant ◽

Content Type ◽

Hospital Acquired Infections ◽

Positive Strain ◽

Hospital Acquired ◽

Norwegian Patient

ABSTRACT Multidrug-resistant Klebsiella pneumoniae is a major cause of hospital-acquired infections. Here, we report the complete genome sequence of the multidrug-resistant, bla NDM-1-positive strain K. pneumoniae K66-45, isolated from a hospitalized Norwegian patient.

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The Small Protein RmpD Drives Hypermucoviscosity in Klebsiella pneumoniae

mBio ◽

10.1128/mbio.01750-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Kimberly A. Walker ◽

Logan P. Treat ◽

Victoria E. Sepúlveda ◽

Virginia L. Miller

Keyword(s):

Gene Expression ◽

Drug Resistance ◽

Klebsiella Pneumoniae ◽

Transmembrane Domain ◽

Transcriptional Regulator ◽

Clinical Isolates ◽

Small Protein ◽

Content Type ◽

Capsule Production ◽

Wide Range

ABSTRACT Klebsiella pneumoniae has a remarkable ability to cause a wide range of human diseases. It is divided into two broad classes: classical strains that are a notable problem in health care settings due to multidrug resistance, and hypervirulent (hv) strains that are historically drug sensitive but able to establish disease in immunocompetent hosts. Alarmingly, there has been an increased frequency of clinical isolates that have both drug resistance and hv-associated genes. One such gene, rmpA, encodes a transcriptional regulator required for maximal capsule (cps) gene expression and confers hypermucoviscosity (HMV). This link has resulted in the assumption that HMV is caused by elevated capsule production. However, we recently reported a new cps regulator, RmpC, and ΔrmpC mutants have reduced cps expression but retain HMV, suggesting that capsule production and HMV may be separable traits. Here, we report the identification of a small protein, RmpD, that is essential for HMV but does not impact capsule. RmpD is 58 residues with a putative N-terminal transmembrane domain and highly positively charged C-terminal half, and it is conserved among other hv K. pneumoniae strains. Expression of rmpD in trans complements both ΔrmpD and ΔrmpA mutants for HMV, suggesting that RmpD is the key driver of this phenotype. The rmpD gene is located between rmpA and rmpC, within an operon regulated by RmpA. These data, combined with our previous work, suggest a model in which the RmpA-associated phenotypes are largely due to RmpA activating the expression of rmpD to produce HMV and rmpC to stimulate cps expression. IMPORTANCE Capsule is a critical virulence factor in Klebsiella pneumoniae, in both antibiotic-resistant classical strains and hypervirulent strains. Hypervirulent strains usually have a hypermucoviscosity (HMV) phenotype that contributes to their heightened virulence capacity, but the production of HMV is not understood. The transcriptional regulator RmpA is required for HMV and also activates capsule gene expression, leading to the assumption that HMV is caused by hyperproduction of capsule. We have identified a new gene (rmpD) required for HMV but not for capsule production. This distinction between HMV and capsule production will promote a better understanding of the mechanisms of hypervirulence, which is in great need given the alarming increase in clinical isolates with both drug resistance and hypervirulence traits.

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Inactivation of Nitrite-Dependent Nitric Oxide Biosynthesis Is Responsible for Overlapped Antibiotic Resistance between Naturally and Artificially Evolved Pseudomonas aeruginosa

mSystems ◽

10.1128/msystems.00732-21 ◽

2021 ◽

Author(s):

Su-fang Kuang ◽

Ding-yun Feng ◽

Zhuang-gui Chen ◽

Zhuo-zheng Liang ◽

Juan-juan Xiang ◽

...

Keyword(s):

Nitric Oxide ◽

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Content Type ◽

Hospital Acquired Infections ◽

Hospital Acquired

Infections with Pseudomonas aeruginosa have become a real concern among hospital-acquired infections, especially in cystic fibrosis patients and immunocompromised individuals. Control of the pathogen is challenging due to antibiotic resistance.

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OXA-48 Carbapenemase in Klebsiella pneumoniae Sequence Type 307 in Ecuador

Microorganisms ◽

10.3390/microorganisms8030435 ◽

2020 ◽

Vol 8 (3) ◽

pp. 435

Author(s):

José E. Villacís ◽

Jorge A. Reyes ◽

Hugo G. Castelán-Sánchez ◽

Sonia Dávila-Ramos ◽

Miguel Angel Lazo ◽

...

Keyword(s):

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Great Increase ◽

Multidrug Resistant ◽

Sequence Type ◽

Negative Bacterium ◽

Molecular Surveillance ◽

Hospital Acquired Infections ◽

First Case ◽

Hospital Acquired

Antibiotic resistance is on the rise, leading to an increase in morbidity and mortality due to infectious diseases. Klebsiella pneumoniae is a Gram-negative bacterium that causes bronchopneumonia, abscesses, urinary tract infection, osteomyelitis, and a wide variety of infections. The ubiquity of this microorganism confounds with the great increase in antibiotic resistance and have bred great concern worldwide. K. pneumoniae sequence type (ST) 307 is a widespread emerging clone associated with hospital-acquired infections, although sporadic community infections have also been reported. The aim of our study is to describe the first case of Klebsiella pneumoniae (ST) 307 harboring the blaOXA-48-like gene in Ecuador. We characterized a new plasmid that carry OXA-48 and could be the source of future outbreaks. The strain was recovered from a patient with cancer previously admitted in a Ukrainian hospital, suggesting that this mechanism of resistance could be imported. These findings highlight the importance of programs based on active molecular surveillance for the intercontinental spread of multidrug-resistant microorganisms with emergent carbapenemases.

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Antagonistic Donor Density Effect Conserved in Multiple Enterococcal Conjugative Plasmids

Applied and Environmental Microbiology ◽

10.1128/aem.00363-16 ◽

2016 ◽

Vol 82 (15) ◽

pp. 4537-4545 ◽

Cited By ~ 13

Author(s):

Arpan Bandyopadhyay ◽

Sofie O'Brien ◽

Kristi L. Frank ◽

Gary M. Dunny ◽

Wei-Shou Hu

Keyword(s):

Antibiotic Resistance ◽

Enterococcus Faecalis ◽

Virulence Determinants ◽

Content Type ◽

Hospital Acquired Infections ◽

Donor Density ◽

Donor Cells ◽

Inhibitor Peptide ◽

Hospital Acquired ◽

High Donor

ABSTRACTEnterococcus faecalis, a common causative agent of hospital-acquired infections, is resistant to many known antibiotics. Its ability to acquire and transfer resistance genes and virulence determinants through conjugative plasmids poses a serious concern for public health. In some cases, induction of transfer ofE. faecalisplasmids results from peptide pheromones produced by plasmid-free recipient cells, which are sensed by the plasmid-bearing donor cells. These plasmids generally encode an inhibitory peptide that competes with the pheromone and suppresses self-induction of donors. We recently demonstrated that the inhibitor peptide encoded on plasmid pCF10 is part of a unique quorum-sensing system in which it functions as a “self-sensing signal,” reducing the response to the pheromone in a density-dependent fashion. Based on the similarities between regulatory features controlling conjugation in pAD1 and pAM373 and those controlling conjugation in pCF10, we hypothesized that these plasmids are likely to exhibit similar quorum-sensing behaviors. Experimental findings indicate that for both pAD1 and pAM373, high donor densities indeed resulted in decreased induction of the conjugation operon and reduced conjugation frequencies. This effect was restored by the addition of exogenous inhibitor, confirming that the inhibitor serves as an indicator for donor density. Donor density also affects cross-species conjugative plasmid transfer. Based on our experimental results, we propose models for induction and shutdown of the conjugation operon in pAD1 and pAM373.IMPORTANCEEnterococcus faecalisis a leading cause of hospital-acquired infections. Its ability to transfer antibiotic resistance and virulence determinants by sharing its genetic material with other bacteria through direct cell-cell contact via conjugation poses a serious threat. Two antagonistic signaling peptides control the transfer of plasmids pAD1 and pAM373: a peptide pheromone produced by plasmid-free recipients triggers the conjugative transfer in plasmid-containing donors, and an inhibitor peptide encoded on the plasmid and produced by donor cells serves to modulate the donor response in accordance with the relative abundance of donors and recipients. We demonstrate that high donor density reduces the conjugation frequency of both of these plasmids, which is a consequence of increased inhibitor concentration in high-donor-density cultures. While most antibiotic strategies end up selecting resistant strains and disrupting the community balance, manipulating bacterial signaling mechanisms can serve as an alternate strategy to prevent the spread of antibiotic resistance.

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