RNA signatures allow rapid identification of pathogens and antibiotic susceptibilities

Barczak, Amy K.; Gómez, James; Kaufmann, B.; Hinson, Ella R.; Cosimi, Lisa A.; Borowsky, Mark; Onderdonk, Andrew B.; Stanley, Sarah A.; Kaur, Devinder; Bryant, Kevin F.; Knipe, David M.; Sloutsky, Alexander; Hung, Deborah T.

doi:10.1073/pnas.1119540109

Cited by 100 publications

(109 citation statements)

References 29 publications

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“…Based on the interaction of Ppr1 with both Rip1-PDZ and RsmA, we hypothesized that Ppr1 may affect activation of the SigM branch of the Rip1 pathway. SigM positively auto-regulates its own transcription (34), and therefore we measured the abundance of the mRNA encoding SigM by two methods, quantitative RT-PCR and NanoString (35), in the Δppr1 strain. By both methods, we observed that the mRNA encoding SigM was overexpressed in Δppr1 cells compared with either WT or Δppr1 complemented cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

Site-2 protease substrate specificity and coupling in trans by a PDZ-substrate adapter protein

Schneider¹,

Reddy²,

E³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Site-2 proteases (S2Ps) are intramembrane metalloproteases that cleave transmembrane substrates in all domains of life. Many S2Ps, including human S2P and Mycobacterium tuberculosis Rip1, have multiple substrates in vivo, which are often transcriptional regulators. However, S2Ps will also cleave transmembrane sequences of nonsubstrate proteins, suggesting additional specificity determinants. Many S2Ps also contain a PDZ domain, the function of which is poorly understood. Here, we identify an M. tuberculosis protein, PDZ-interacting protease regulator 1 (Ppr1), which bridges between the Rip1 PDZ domain and anti-sigma factor M (Anti-SigM), a Rip1 substrate, but not Anti-SigK or Anti-SigL, also Rip1 substrates. In vivo analyses of Ppr1 function indicate that it prevents nonspecific activation of the Rip1 pathway while coupling Rip1 cleavage of Anti-SigM, but not Anti-SigL, to site-1 proteolysis. Our results support a model of S2P substrate specificity in which a substratespecific adapter protein tethers the S2P to its substrate while holding the protease inactive through its PDZ domain.intramembrane proteolysis | signal transduction | Tuberculosis | Sigma factor R egulated intramembrane proteolysis (RIP) is a widely distributed mechanism of signal transduction across membranes in which specialized intramembrane cleaving proteases (iCLIPs) cleave the transmembrane segments of substrate proteins (1, 2). The site-2 (S2) proteases (S2Ps) are one class of iCLIP that is widely distributed from bacteria to human cells and participate in such diverse pathways as lipid biosynthesis (1, 3, 4), sporulation (5), membrane stress (6, 7), alginate production (8), and polar morphogenesis (9). In bacteria, PDZ domain containing S2Ps often control the release of extracytoplasmic function sigma factors from the membrane through proteolysis of their cognate membrane-bound anti-sigma factors (10, 11). S2Ps are so named because the intramembrane cleavage event follows cleavage by a site-1 (S1) protease (S1P). The S1P first cleaves the periplasmic domain of the anti-sigma factor in response to an extracellular signal. Only after S1 cleavage does the S2P cleave near the cytoplasmic side of the transmembrane domain of the anti-sigma factor, liberating the anti-sigma/sigma factor complex from the membrane into the cytoplasm where the sigma factor can associate with RNA polymerase. A specific extracytoplasmic stimulus can thus be coupled to a transcriptional response through a coordinated proteolytic cascade.One of the major unanswered questions in S2P-mediated RIP is how these proteases achieve specificity in signaling. In many cases, S2Ps have multiple substrates, including human S2P, which cleaves SREBPs, ATF6, and CREB-H (12, 13); Bacillus subtilis RasP, which cleaves both RsiW as well as FtsL (14, 15); and Mycobacterium tuberculosis regulated intramembrane protease 1 (Rip1), which cleaves three anti-sigma factors, Anti-SigK (RskA), Anti-SigL (RslA), and Anti-SigM (RsmA) (3), as well as PBP3 (16). However, there is substantial evidence t...

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Section: Resultsmentioning

confidence: 99%

Site-2 protease substrate specificity and coupling in trans by a PDZ-substrate adapter protein

Schneider¹,

Reddy²,

E³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…In the present study, M. tuberculosis cultures treated with INH or RIF had rapid decreases in Ag85B mRNA and accurately reported drug susceptibility and resistance (15). Also, multiplexed quantitative detection of several M. tuberculosis mRNA targets using nCounter analysis (Nanostring Technologies) accurately reports phenotypic AST of cultured clinical strains (16). To our knowledge, the effectiveness of either of these approaches has not yet been validated using clinical samples, perhaps due to the complex mixture of viable, dying, and dead cells present in sputum samples that may complicate the relationship between drug effects and cellular mRNA turnover.…”

Section: Discussionmentioning

confidence: 99%

Optimization of a Nucleic Acid-Based Reporter System To Detect Mycobacterium tuberculosis Antibiotic Sensitivity

Mulvey

Lemmon

Rotter

et al. 2015

Antimicrob Agents Chemother

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bWe previously reported the development of a prototype antibiotic sensitivity assay to detect drug-resistant Mycobacterium tuberculosis using infection by mycobacteriophage to create a novel nucleic acid transcript, a surrogate marker of mycobacterial viability, detected by reverse transcriptase PCR (M. C. Mulvey et al., mBio 3:e00312-11, 2012). This assay detects antibiotic resistance to all drugs, even drugs for which the resistance mechanism is unknown or complex: it is a phenotypic readout using nucleic acid detection. In this report, we describe development and characteristics of an optimized reporter system that directed expression of the RNA cyclase ribozyme, which generated circular RNA through an intramolecular splicing reaction and led to accumulation of a new nucleic acid sequence in phage-infected bacteria. These modifications simplified the assay, increased the limit of detection from 10 4 to <10 2 M. tuberculosis cells, and correctly identified the susceptibility profile of M. tuberculosis strains exposed for 16 h to either first-line or second-line antitubercular drugs. In addition to phenotypic drug resistance or susceptibility, the assay reported streptomycin MICs and clearly detected 10% drug-resistant cells in an otherwise drug-susceptible population. Multidrug-resistant tuberculosis (MDR-TB) is caused by Mycobacterium tuberculosis strains resistant to at least two of the drugs, isoniazid (INH) and rifampin (RIF), in the recommended four-drug TB treatment regimen (1). The World Health Organization (WHO) estimates the prevalence of MDR-TB to be approximately 50 million people worldwide (2), expanding by nearly 500,000 new cases each year (3). A more disquieting development is the increase in and global distribution of extremely drug-resistant TB (XDR-TB) (M. tuberculosis resistant to INH, RIF, and key second-line drugs) (4). Early recognition of M/XDR-TB through the use of an accurate and sensitive antibiotic susceptibility test (AST) promises to improve patient outcomes and assist in TB control efforts.Due to the notoriously slow doubling time of M. tuberculosis, a standard AST using culture takes several weeks to months. Culture-based assays are the gold standard for AST, however, because they measure every type of drug susceptibility and do not require knowledge of the genetics underlying resistance. However, they are slow or too insensitive to interrogate effects of antibiotics on small numbers of M. tuberculosis present in most clinical samples. Rapid molecular diagnostic tests such as Xpert MTB/RIF (Cepheid) and GenoType MTBDR (Hain Lifescience) are the emerging standard for rapid INH-or RIF-resistant M. tuberculosis detection, markers of MDR-TB, because their detection thresholds from patient specimens are comparable to culture (5, 6). These tests identify M. tuberculosis DNA in a patient sample and detect common mutations that confer resistance to INH (GenoType MTBDR) and/or RIF (GenoType MTBDR and Xpert MTB/RIF) in 2 h (Xpert MTB/RIF) or 2 days (GenoType MTBDR) from the initiation of t...

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“…Therefore, an ideal monitoring assay should not only identify the presence of bla NDM-1 but also reveal functional bla NDM-1 variants. In view of these requirements, the current widely used PCR sequencing method is not sufficient, because phenotypic testing (expression of recombinant protein) is needed to confirm whether a new variant identified by sequencing has an alteration in the function of its coded protein (14,31).…”

Section: Discussionmentioning

confidence: 99%

Rapid Detection of New Delhi Metallo-β-Lactamase Gene and Variants Coding for Carbapenemases with Different Activities by Use of a PCR-Based In Vitro Protein Expression Method

Huang

Zhou

et al. 2014

J Clin Microbiol

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New Delhi metallo-␤-lactamase (NDM)-producing bacteria are considered potential global health threats. It is necessary to monitor NDM-1 and its variants in clinical isolates in order to understand the NDM-1 epidemic and the impact of its variants on ␤-lactam resistance. To reduce the lengthy time needed for cloning and expression of NDM-1 variants, a novel PCR-based in vitro protein expression (PCR-P) method was used to detect bla NDM-1 and its variants coding for carbapenemases with different activities (functional variants). The PCR-P method combined a long-fragment real-time quantitative PCR (LF-qPCR) with in vitro cell-free expression to convert the bla NDM-1 amplicons into NDM for carbapenemase assay. The method could screen for bla NDM-1 within 3 h with a detection limit of 5 copies and identify functional variants within 1 day. Using the PCR-P to analyze 5 recent bla NDM-1 variants, 2 functional variants, bla NDM-4 and bla NDM-5 , were revealed. In the initial testing of 23 clinical isolates, the PCR-P assay correctly found 8 isolates containing bla NDM-1 . This novel method provides the first integrated approach for rapidly detecting the full-length bla NDM-1 and revealing its functional variants in clinical isolates.

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RNA signatures allow rapid identification of pathogens and antibiotic susceptibilities

Cited by 100 publications

References 29 publications

Site-2 protease substrate specificity and coupling in trans by a PDZ-substrate adapter protein

Site-2 protease substrate specificity and coupling in trans by a PDZ-substrate adapter protein

Optimization of a Nucleic Acid-Based Reporter System To Detect Mycobacterium tuberculosis Antibiotic Sensitivity

Rapid Detection of New Delhi Metallo-β-Lactamase Gene and Variants Coding for Carbapenemases with Different Activities by Use of a PCR-Based In Vitro Protein Expression Method

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