Genome analysis identifies a spontaneous nonsense mutation in ppsD leading to attenuation of virulence in laboratory-manipulated Mycobacterium tuberculosis.

BACKGROUND: A previous laboratory study involving wild type, mutant and devR/dosR complemented strains of Mycobacterium tuberculosis reported the attenuation phenotype of complemented strain, Comp1. This phenotype was intriguing since the parental strain H37Rv, devR mutant (Mut1) and additional complemented strains, Comp9 and Comp11, were virulent in the guinea pig model. RESULTS: Towards deciphering the mechanism underlying the attenuation of Comp1, a whole genome sequencing approach was undertaken. Eight Single Nucleotide Polymorphisms (SNPs) unique to the Comp1 strain were identified. Of these, 5 SNPs were non-synonymous and included a G➞A mutation resulting in a W1591Stop mutation in ppsD gene of the phthiocerol dimycocerosate (PDIM) biosynthetic cluster. Targeted sequence analysis confirmed this mutation in only Comp1 strain and not in wild type (H37Rv), devR knockout (Mut1) or other complemented (Comp9 and Comp11) bacteria. Differential expression of the PDIM locus in Comp1 bacteria was observed which was associated with a partial deficiency of PDIM, an increased sensitivity to detergent and a compromised ability to infect human THP-1 cells. CONCLUSIONS: It is proposed that a spontaneous mutation in the ppsD gene of Comp1 underlies down-modulation of the PDIM locus which is associated with defects in permeability and infectivity as well as virulence attenuation in guinea pigs. Our study demonstrates the value of whole genome sequencing for resolving unexplainable bacterial phenotypes and recommends the assessment of PDIM status while assessing virulence properties of laboratory-manipulated strains of M. tuberculosis.

Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders.

CRISPR/Cas9 holds immense potential to treat a range of genetic disorders. Allele-specific gene disruption induced by non-homologous end-joining (NHEJ) DNA repair offers a potential treatment option for autosomal dominant disease. Here, we successfully delivered a plasmid encoding S. pyogenes Cas9 and sgRNA to the corneal epithelium by intrastromal injection and acheived long-term knockdown of a corneal epithelial reporter gene, demonstrating gene disruption via NHEJ in vivo. In addition, we used TGFBI corneal dystrophies as a model of autosomal dominant disease to assess the use of CRISPR/Cas9 in two allele-specific systems, comparing cleavage using a SNP-derived PAM to a guide specific approach. In vitro, cleavage via a SNP-derived PAM was found to confer stringent allele-specific cleavage, while a guide-specific approach lacked the ability to distinguish between the wild-type and mutant alleles. The failings of the guide-specific approach highlights the necessity for meticulous guide design and assessment, as various degrees of allele-specificity are achieved depending on the guide sequence employed. A major concern for the use of CRISPR/Cas9 is its tendency to cleave DNA non-specifically at "off-target" sites. Confirmation that S. pyogenes Cas9 lacks the specificity to discriminate between alleles differing by a single base-pair regardless of the position in the guide is demonstrated.

Correction: Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation.

[This corrects the article DOI: 10.1371/journal.ppat.1004627.].

Elucidation of the RamA regulon in Klebsiella pneumoniae reveals a role in LPS regulation.

Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins.

Molecular basis of non-mutational derepression of ramA in Klebsiella pneumoniae.

OBJECTIVES: The ram locus, consisting of the romA-ramA genes, is repressed by the tetracycline-type regulator RamR, where regulation is abolished due to loss-of-function mutations within the protein or ligand interactions. The aim of this study was to determine whether the phenothiazines (chlorpromazine and thioridazine) directly interact with RamR to derepress ramA expression. METHODS: Quantitative real-time PCR analyses were performed to determine expression levels of the romA-ramA genes after exposure to the phenothiazines. Electrophoretic mobility shift assays (EMSAs) and in vitro transcription experiments were performed to show direct binding to and repression by RamR. Direct binding of the RamR protein to the phenothiazines was measured by fluorescence spectroscopy experiments and molecular docking models were generated using the RamR crystal structure. RESULTS: Exposure to either chlorpromazine or thioridazine resulted in the up-regulation of the romA-ramA genes. EMSAs and in vitro transcription experiments demonstrated that both agents reduce/abolish binding and enhance transcription of the target PI promoter upstream of the ramR-romA genes in Klebsiella pneumoniae compared with RamR alone. Fluorescence spectroscopy measurements demonstrated that RamR directly binds both chlorpromazine and thioridazine with micromolar affinity. Molecular docking analyses using the RamR crystal structure demonstrated that the phenothiazines interact with RamR protein through contacts described for other ligands, in addition to forming unique strong polar interactions at positions D152 and K63. CONCLUSIONS: These data demonstrate that phenothiazines can modulate loci linked to the microbe-drug response where RamR is an intracellular target for the phenothiazines, thus resulting in a transient non-mutational derepression of ramA concentrations.

Genome Sequence of Klebsiella pneumoniae Ecl8, a Reference Strain for Targeted Genetic Manipulation.

We report the genome sequence of Klebsiella pneumoniae subsp. pneumoniae Ecl8, a spontaneous streptomycin-resistant mutant of strain ECL4, derived from NCIB 418. K. pneumoniae Ecl8 has been shown to be genetically tractable for targeted gene deletion strategies and so provides a platform for in-depth analyses of this species.

Genetic characterization of tigecycline resistance in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes.

OBJECTIVES: The intrinsically encoded ramA gene has been linked to tigecycline resistance through the up-regulation of efflux pump AcrAB in Enterobacter cloacae. The molecular basis for increased ramA expression in E. cloacae and Enterobacter aerogenes, as well as the role of AraC regulator rarA, has not yet been shown. To ascertain the intrinsic molecular mechanism(s) involved in tigecycline resistance in Enterobacter spp., we analysed the expression levels of ramA and rarA and corresponding efflux pump genes acrAB and oqxAB in Enterobacter spp. clinical isolates. METHODS: The expression levels of ramA, rarA, oqxA and acrA were tested by quantitative real-time RT-PCR. The ramR open reading frames of the ramA-overexpressing strains were sequenced; strains harbouring mutations were transformed with wild-type ramR to study altered ramA expression and tigecycline susceptibility. RESULTS: Tigecycline resistance was mediated primarily by increased ramA expression in E. cloacae and E. aerogenes. Only the ramA-overexpressing E. cloacae isolates showed increased rarA and oqxA expression. Upon complementation with wild-type ramR, all Enterobacter spp. containing ramR mutations exhibited decreased ramA and acrA expression and increased tigecycline susceptibility. Exceptions were one E. cloacae strain and one E. aerogenes strain, where a decrease in ramA levels was not accompanied by lower acrA expression. CONCLUSIONS: Increased ramA expression due to ramR deregulation is the primary mediator of tigecycline resistance in clinical isolates of E. cloacae and E. aerogenes. However, some ramA-overexpressing isolates do not show changes in ramR, suggesting alternate pathways of ramA regulation; the rarA regulator and the oqxAB efflux pump may also play a role in tigecycline resistance in E. cloacae.

Elucidating the regulon of multidrug resistance regulator RarA in Klebsiella pneumoniae.

RarA is an AraC-type regulator in Klebsiella pneumoniae, which, when overexpressed, confers a low-level multidrug-resistant (MDR) phenotype linked to the upregulation of both the acrAB and oqxAB efflux genes. Increased rarA expression has also been shown to be integral in the development of tigecycline resistance in the absence of ramA in K. pneumoniae. Given its phenotypic role in MDR, microarray analyses were performed to determine the RarA regulon. Transcriptome analysis was undertaken using strains Ecl8ΔrarA/pACrarA-2 (rarA-expressing construct) and Ecl8ΔrarA/pACYC184 (vector-only control) using bespoke microarray slides consisting of probes derived from the genomic sequences of K. pneumoniae MGH 78578 (NC_009648.1) and Kp342 (NC_011283.1). Our results show that rarA overexpression resulted in the differential expression of 66 genes (42 upregulated and 24 downregulated). Under the COG (clusters of orthologous groups) functional classification, the majority of affected genes belonged to the category of cell envelope biogenesis and posttranslational modification, along with genes encoding the previously uncharacterized transport proteins (e.g., KPN_03141, sdaCB, and leuE) and the porin OmpF. However, genes associated with energy production and conversion and amino acid transport/metabolism (e.g., nuoA, narJ, and proWX) were found to be downregulated. Biolog phenotype analyses demonstrated that rarA overexpression confers enhanced growth of the overexpresser in the presence of several antibiotic classes (i.e., beta-lactams and fluoroquinolones), the antifungal/antiprotozoal compound clioquinol, disinfectants (8-hydroxyquinoline), protein synthesis inhibitors (i.e., minocycline and puromycin), membrane biogenesis agents (polymyxin B and amitriptyline), DNA synthesis (furaltadone), and the cytokinesis inhibitor (sanguinarine). Both our transcriptome and phenotypic microarray data support and extend the role of RarA in the MDR phenotype of K. pneumoniae.

Simplified detection of Mycobacterium tuberculosis in sputum using smear microscopy and PCR with molecular beacons.

The prompt diagnosis of smear-negative cases is a prerequisite to controlling tuberculosis (TB). Several new laboratory approaches, including nucleic acid amplification (NAA), are being evaluated in various disease settings to meet this challenge. However, NAA needs simplification before it is widely accepted. Furthermore, a supporting smear result improves confidence in and reliability of PCR. In this context, an asymmetric devR PCR assay using two molecular beacon probes for visual or fluorimetric end-point detection of Mycobacterium tuberculosis was developed. The assays reproducibly detected 25 fg M. tuberculosis DNA versus 100 fg by conventional gel electrophoresis (henceforth referred to as gel assay). The devR and IS6110 PCR assays were blindly evaluated on sputum specimens obtained from a directly observed-treatment short-course centre. Universal sample processing (USP) smear microscopy and culture were used as a supportive test and the 'gold' standard, respectively. Among the 148 specimens analysed, 120 were M. tuberculosis culture-positive. Amongst the 122 direct smear-negative samples, 96 were culture-positive, of which 61 were detected by USP smear microscopy. All 35 USP smear-negative samples were positive by three or more PCR methods. devR PCR had a sensitivity of 92.5 % in the fluorimetric assay versus 86.7 % by visual inspection and 90.8 % by the gel method. IS6110 PCR performed at almost equivalent levels. devR visual and fluorimetric assays considered together yielded an increased sensitivity of 95 % without compromising on a specificity of 92.9 %. The results suggest that the USP smear test is useful for diagnosing direct smear-negative TB and judiciously restricting PCR testing to only smear-negative samples. When used together, these tests can provide rapid diagnosis of smear-negative TB in a cost-effective manner.