Next-generation Diagnostics for Melioidosis: Evaluation of a Prototype i-STAT Cartridge to Detect Burkholderia pseudomallei Biomarkers.

BACKGROUND: Infection with the gram-negative bacterium Burkholderia pseudomallei can result in melioidosis, a life-threatening disease that can be difficult to diagnose. Culture remains the gold standard for diagnosis but requires laboratory resources not available in many endemic regions. A lateral flow immunoassay has shown promise for POC diagnostics but suffers from low sensitivity when used on blood samples. PCR also has low sensitivity on blood, attributed to the low bacterial numbers in blood observed in melioidosis patients, even when bacteraemic. METHODS: A prototype i-STAT cartridge was developed to utilize the monoclonal antibody specific for the capsule of pathogenic Burkholderia species employed on the LFI. The resulting POC assay was evaluated on 414 clinical specimens from Darwin, Australia and Cambodia. RESULTS: The i-STAT assay accurately distinguished Australian blood culture positive melioidosis patients from Australian patients hospitalized with other infections (AUC = 0.91, 95% CI 0.817 - 1.0). We derived an assay cutoff with 76% sensitivity and 94% specificity that correctly classified 88% (n = 74) of the Australian patients. Interestingly, only 46% (6/13) of the culture-positive melioidosis patients in Cambodia were classified correctly. Of great importance however, the assay detected capsule from blood samples for 32% of blood culture negative melioidosis patients in both cohorts and previously undiagnosed melioidosis patients in Cambodia. In addition the assay showed high sensitivity and specificity for urine, pus and sputum. CONCLUSIONS: Diagnostic tools that are not dependent upon the growth kinetics or the levels of bacteremia of B. pseudomallei represent the next-generation of diagnostics and must be pursued further.

Target prediction for small, noncoding RNAs in bacteria.

Many small, noncoding RNAs in bacteria act as post-transcriptional regulators by basepairing with target mRNAs. While the number of characterized small RNAs (sRNAs) has steadily increased, only a limited number of the corresponding mRNA targets have been identified. Here we present a program, TargetRNA, that predicts the targets of these bacterial RNA regulators. The program was evaluated by assessing whether previously known targets could be identified. The program was then used to predict targets for the Escherichia coli RNAs RyhB, OmrA, OmrB and OxyS, and the predictions were compared with changes in whole genome expression patterns observed upon expression of the sRNAs. Our results show that TargetRNA is a useful tool for finding mRNA targets of sRNAs, although its rate of success varies between sRNAs.

Controlling mRNA stability and translation with small, noncoding RNAs.

Recent studies have led to the identification of more than 50 small regulatory RNAs in Escherichia coli. Only a subset of these RNAs has been characterized. However, it is clear that many of the RNAs, such as the MicF, OxyS, DsrA, Spot42 and RyhB RNAs, act by basepairing to activate or repress translation or to destabilize mRNAs. Basepairing between these regulatory RNAs and their target mRNAs requires the Sm-like Hfq protein which most likely functions as an RNA chaperone to increase RNA unfolding or local target RNA concentration. Here we summarize the physiological roles of the basepairing RNAs, examine their prevalence in bacteria and discuss unresolved questions regarding their mechanisms of action.

RNase III participates in GadY-dependent cleavage of the gadX-gadW mRNA.

The adjacent gadX and gadW genes encode transcription regulators that are part of a complex regulatory circuit controlling the Escherichia coli response to acid stress. We previously showed that the small RNA GadY positively regulates gadX mRNA levels. The gadY gene is located directly downstream of the gadX coding sequence on the opposite strand of the chromosome. We now report that gadX is transcribed in an operon with gadW, although this full-length mRNA does not accumulate. Base pairing of the GadY small RNA with the intergenic region of the gadX-gadW mRNA results in directed processing events within the region of complementarity. The resulting two halves of the cleaved mRNA accumulate to much higher levels than the unprocessed mRNA. We examined the ribonucleases required for this processing, and found that multiple enzymes are involved in the GadY-directed cleavage including the double-strand RNA-specific endoribonuclease RNase III.

Mutational analysis to define an activating region on the redox-sensitive transcriptional regulator OxyR.

The OxyR transcription factor is a key regulator of the Escherichia coli response to oxidative stress. Previous studies showed that OxyR binding to a target promoter enhances RNA polymerase binding and vice versa, suggesting a direct interaction between OxyR and RNA polymerase. To identify the region of OxyR that might contact RNA polymerase, we carried out alanine scanning and random mutagenesis of oxyR. The combination of these approaches led to the identification of several mutants defective in the activation of an OxyR target gene. A subset of the mutations map to the DNA-binding domain, other mutations appear to affect dimerization of the regulatory domain, while another group is suggested to affect disulfide bond formation. The two mutations, D142A and R273H, giving the most dramatic phenotype are located in a patch on the surface of the oxidized OxyR protein and possibly define an activating region on OxyR.

GadY, a small-RNA regulator of acid response genes in Escherichia coli.

A previous bioinformatics-based search for small RNAs in Escherichia coli identified a novel RNA named IS183. The gene encoding this small RNA is located between and on the opposite strand of genes encoding two transcriptional regulators of the acid response, gadX (yhiX) and gadW (yhiW). Given that IS183 is encoded in the gad gene cluster and because of its role in regulating acid response genes reported here, this RNA has been renamed GadY. We show that GadY exists in three forms, a long form consisting of 105 nucleotides and two processed forms, consisting of 90 and 59 nucleotides. The expression of this small RNA is highly induced during stationary phase in a manner that is dependent on the alternative sigma factor sigmaS. Overexpression of the three GadY RNA forms resulted in increased levels of the mRNA encoding the GadX transcriptional activator, which in turn caused increased levels of the GadA and GadB glutamate decarboxylases. A promoter mutation which abolished gadY expression resulted in a reduction in the amount of gadX mRNA during stationary phase. The gadY gene was shown to overlap the 3' end of the gadX gene, and this overlap region was found to be necessary for the GadY-dependent accumulation of gadX mRNA. We suggest that during stationary phase, GadY forms base pairs with the 3'-untranslated region of the gadX mRNA and confers increased stability, allowing for gadX mRNA accumulation and the increased expression of downstream acid resistance genes.

Expression of the secondary sigma factor sigmaX in Streptococcus pyogenes is restricted at two levels.

Secondary RNA polymerase sigma factors in many bacteria are responsible for regulating a vast range of processes including virulence. A protein (sigma(X)) in the gram-positive human pathogen Streptococcus pyogenes (the group A Streptococcus or GAS) was recently shown to function in vitro as a secondary sigma factor. We report here the isolation of a mutant in which both sigX genes are inactivated, show that sigma(X) functions in GAS cells, and show that the amount of sigma(X) is controlled at two levels. Primer extension analysis indicates that sigX transcription is low in GAS cells grown in Todd-Hewitt yeast broth, and immunoblot assays with a sigma(X)-specific polyclonal antibody demonstrate that the protein does not accumulate in these cells. To increase the level of sigX transcription in GAS, we constructed a strain that constitutively expresses the sigX gene from a heterologous promoter. Expression of sigX from this promoter led to transcription of the sigma(X)-dependent cinA promoter in GAS cells. We found that expression of the sigX gene in a clpP mutant strain resulted in greater accumulation of sigma(X) protein, which resulted in higher levels of transcription from the sigma(X)-dependent promoters cinA, smf, and cglA. In addition, a clpP mutant containing sigX only at its wild-type loci on the chromosome generated more transcription from the sigma(X)-dependent cinA promoter than did the wild-type parental strain. Therefore, sigma(X) activity in GAS is limited by low-level transcription of the sigX structural genes and by clpP, which appears to negatively regulate sigma(X) accumulation.