Dual role of RsmA in the coordinated regulation of expression of virulence genes in Pectobacterium wasabiae strain SCC3193.

The CsrA/RsmA family of post-transcriptional regulators in bacteria is involved in regulating many cellular processes, including pathogenesis. Using a bioinformatics approach, we identified an RsmA binding motif, A(N)GGA, in the Shine-Dalgarno regions of 901 genes. Among these genes with the predicted RsmA binding motif, 358 were regulated by RsmA according to our previously published gene expression profiling analysis (WT vs rsmA negative mutant; Kõiv et al., 2013). A small subset of the predicted targets known to be important as virulence factors was selected for experimental validation. RNA footprint analyses demonstrated that RsmA binds specifically to the ANGGA motif in the 5'UTR sequences of celV1, pehA, pelB, pel2 and prtW. RsmA-dependent regulation of these five genes was examined in vivo using plasmid-borne translational and transcriptional fusions with a reporter gusA gene. They were all affected negatively by RsmA. However, we demonstrated that whereas the overall effect of RsmA on celV1 and prtW was determined on both the translational and transcriptional level, expression of pectinolytic enzyme genes (pehA, pel2 and pelB) was affected mainly on the level of transcription in tested conditions. In summary, these data indicate that RsmA controls virulence by integration of its regulatory activities at various levels.

Sensitive and rapid detection of Chlamydia trachomatis by recombinase polymerase amplification directly from urine samples.

Chlamydia trachomatis is the most common sexually transmitted human pathogen. Infection results in minimal to no symptoms in approximately two-thirds of women and therefore often goes undiagnosed. C. trachomatis infections are a major public health concern because of the potential severe long-term consequences, including an increased risk of ectopic pregnancy, chronic pelvic pain, and infertility. To date, several point-of-care tests have been developed for C. trachomatis diagnostics. Although many of them are fast and specific, they lack the required sensitivity for large-scale application. We describe a rapid and sensitive form of detection directly from urine samples. The assay uses recombinase polymerase amplification and has a minimum detection limit of 5 to 12 pathogens per test. Furthermore, it enables detection within 20 minutes directly from urine samples without DNA purification before the amplification reaction. Initial analysis of the assay from clinical patient samples had a specificity of 100% (95% CI, 92%-100%) and a sensitivity of 83% (95% CI, 51%-97%). The whole procedure is fairly simple and does not require specific machinery, making it potentially applicable in point-of-care settings.

Lack of RsmA-mediated control results in constant hypervirulence, cell elongation, and hyperflagellation in Pectobacterium wasabiae.

The posttranscriptional regulator RsmA controls the production of plant cell wall degrading enzymes (PCWDE) and cell motility in the Pectobacterium genus of plant pathogens. In this study the physiological role of gene regulation by RsmA is under investigation. Disruption of rsmA gene of the Pectobacterium wasabiae strain, SCC3193 resulted in 3-fold decrease in growth rate and increased virulence. The comparison of mRNA levels of the rsmA(-) mutant and wild-type using a genome-wide microarray showed, that genes responsible for successful infection, i.e. virulence factors, motility, butanediol fermentation, various secretion systems etc. were up-regulated in the rsmA(-) strain. The rsmA(-) strain exhibited a higher propensity to swarm and produce PCWDE compared to the wild-type strain. Virulence experiments in potato tubers demonstrated that in spite of its more efficient tissue maceration, the rsmA(-) strain's ability to survive within the host is reduced and the infection site is taken over by resident bacteria. Taken together, in the absence of RsmA, cells revert to a constitutively infective phenotype characterized by expression of virulence factors and swarming. We hypothesize that lack of control over these costly energetic processes results in decreased growth rate and fitness. In addition, our findings suggest a relationship between swarming and virulence in plant pathogens.