Biology subjects

Miller, V. L.

Publications and source records attributed to Miller, V. L..

5 recordsLinked to original sources

bioRxiv2025

The acquisition of rmpADC can increase virulence of classical Klebsiella pneumoniae in the absence of other hypervirulence-associated genes

Klebsiella pneumoniae is one of the most common causes of nosocomial infections, and the rise of drug-resistant K. pneumoniae strains is complicating treatment and contributing to a mounting global health crisis. K. pneumoniae has two pathotypes, classical (cKp) and hypervirulent (hvKp). CKp typically cause opportunistic infections in immunocompromised individuals in healthcare settings and often are multi-drug resistant. HvKp can be community acquired and cause high-mortality infections in immunocompetent individuals. Concerningly, antibiotic-resistant cKp strains with hypervirulence-associated genes and traits have recently emerged. Determining if and how hv-associated genes contribute to increased virulence of cKp strains is essential to addressing this growing threat. The rmpADC operon is an hv-associated locus that confers hypermucoviscosity (HMV), a key virulence phenotype, and rmp genes are often found in convergent strains. In this study, we aimed to determine if the rmp genes alone could increase the virulence of classical strains in the absence of other hv-associated genes. We introduced genetically distinct rmp loci from different lineages into a broad array of cKp isolates and found that, while many isolates became HMV positive, only a subset of these strains showed an increase in virulence in a mouse model of pneumonia. Sequence type and capsule type were not predictive of how rmp acquisition impacted the clinical isolates. Our results indicate that HMV is likely necessary but not sufficient for hypervirulence and that rmp sequence can influence virulence potential in classical strains. IMPORTANCEKlebsiella pneumoniae is a global pathogen, and gene exchange between hypervirulent (hvKp) and classical (cKp) strains is a rising threat. It is essential to understand how hvKp genes impact virulence phenotypes and identify the cKp strain backgrounds most amenable to enhanced virulence. Hypermucoviscosity (HMV) is a critical virulence factor in hypervirulent K. pneumoniae, conferred by the rmpADC locus. The rmp genes are encoded on mobile genetic elements and have been detected in convergent antibiotic-resistant K. pneumoniae strains of concern. In this study, we explored the impact of rmp acquisition in a broad set of classical clinical isolates. We observed that HMV appears necessary, but not sufficient, for increased virulence. Sequence type, capsule type, and HMV capacity could not predict which classical isolates gain an rmp-dependent colonization benefit. These insights increase our understanding of the distinctions between cKp and hvKp and further our ability to identify and treat new strains of concern.

microbiology↗

bioRxiv2024

Genomic and functional analysis of rmp locus variants in Klebsiella pneumoniae

AbstractO_ST_ABSBackgroundC_ST_ABSKlebsiella pneumoniae is an opportunistic pathogen and a leading cause of healthcare-associated infections in hospitals, which are frequently antimicrobial resistant (AMR). Exacerbating the public health threat posed by K. pneumoniae, some strains also harbor additional hypervirulence determinants typically acquired via mobile genetic elements such as the well-characterised large virulence plasmid KpVP-1. The rmpADC locus is considered a key virulence feature of K. pneumoniae and is associated with upregulated capsule expression and the hypermucoid phenotype, which can enhance virulence by contributing to serum resistance. Typically such strains have been susceptible to all antimicrobials besides ampicillin, however the recent emergence of AMR hypermucoid strains is concerning. MethodsHere, we investigate the genetic diversity, evolution, mobilisation and prevalence of rmpADC, in a dataset of 14000 genomes from isolates of the Klebsiella pneumoniae species complex, and describe the RmST virulence typing scheme for tracking rmpADC variants for the purposes of genomic surveillance. Additionally, we examine the functionality of representatives for variants of rmpADC introduced into a mutant strain lacking its native rmpADC locus. ResultsThe rmpADC locus was detected in 7% of the dataset, mostly from genomes of K. pneumoniae and a very small number of K. variicola and K. quasipneumoniae. Sequence variants of rmpADC grouped into five distinct lineages (rmp1, rmp2, rmp2A, rmp3 and rmp4) that corresponded to unique mobile elements, and were differentially distributed across different populations (i.e. clonal groups) of K. pneumoniae. All variants were demonstrated to produce enhanced capsule production and hypermucoviscosity. ConclusionThese results provide an overview of the diversity and evolution of a prominent K. pneumoniae virulence factor and support the idea that screening for rmpADC in K. pneumoniae isolates and genomes is valuable to monitor the emergence and spread of hypermucoid K. pneumoniae, including AMR strains.

genomics↗

bioRxiv2020

The small protein RmpD drives hypermucoviscosity in Klebsiella pneumoniae

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 healthcare settings due to multidrug resistance, and hypervirulent (hv) strains that are 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 is rmpA that 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 {Delta}rmpC mutants have reduced cps expression but remain HMV, suggesting that capsule 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 {Delta}rmpD and {Delta}rmpA 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. This data, combined with our previous work, suggests 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. SIGNIFICANCECapsule is a critical virulence factor in Klebsiella pneumoniae, in both antibiotic-resistant classical strains and hypervirulent strains. Hypervirulent strains usually have a hypermucoviscous (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 does not contribute to 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.

microbiology↗

bioRxiv2018

The Evolution of SlyA/RovA Transcription Factors From Repressors to Counter-Silencers in Enterobacteriaceae

Gene duplication and subsequent evolutionary divergence have allowed conserved proteins to develop unique roles. The MarR family of transcription factors (TFs) has undergone extensive duplication and diversification in bacteria, where they act as environmentally-responsive repressors of genes encoding efflux pumps that confer resistance to xenobiotics, including many antimicrobial agents. We have performed structural, functional, and genetic analyses of representative members of the SlyA/RovA lineage of MarR TFs, which retain some ancestral functions, including repression of their own expression and that of divergently-transcribed multidrug efflux pumps, as well as allosteric inhibition by aromatic carboxylate compounds. However, SlyA and RovA have acquired the ability to counter-silence horizontally-acquired genes, which has greatly facilitated the evolution of Enterobacteriaceae by horizontal gene transfer. SlyA/RovA TFs in different species have independently evolved novel regulatory circuits to provide the enhanced levels of expression required for their new role. Moreover, in contrast to MarR, SlyA is not responsive to copper. These observations demonstrate the ability of TFs to acquire new functions as a result of evolutionary divergence of both cis-regulatory sequences and in trans interactions with modulatory ligands.

microbiology↗

bioRxiv2018

Analysis of novel domain-specific mutations in the zebrafish ndr2/cyclops gene induced by CRISPR-Cas9 RNPs

Nodal-related protein (ndr2) is a member of the transforming growth factor type {beta} superfamily of factors and is required for ventral midline patterning of the embryonic central nervous system in zebrafish. In humans, mutations in the gene encoding nodal cause holoprosencephaly and heterotaxy. Mutations in the ndr2 gene in the zebrafish (Danio rerio) lead to similar phenotypes, including loss of the medial floor plate, severe deficits in ventral forebrain development, and cyclopia. Alleles of the ndr2 gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen different ndr2 alleles have been reported in zebrafish, of which eight were generated using chemical mutagenesis, four were radiation-induced, and the remaining alleles were obtained via random insertion, gene targeting (TALEN), or unknown methods. Therefore, most mutation sites were random and could not be predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of zebrafish ndr2 and observed cyclopia in the injected (G0) embryos. We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (G0) embryos. Targeted PCR amplicon analysis using Sanger sequencing showed that most of the alleles had small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using an in vitro nuclease assay and in vivo analysis using embryos. We describe one specific mutant allele resulting in loss of conserved terminal cysteine-coding sequences. This study is another demonstration of the utility of the CRISPR-Cas9 system in generating domain- specific mutations and provides further insights into the structure-function of the ndr2 gene.

genetics↗