Biology subjects

Tanner, N.

Publications and source records attributed to Tanner, N..

4 recordsLinked to original sources

bioRxiv2021

The transcription factor IscR promotes Yersinia type III secretion system activity by antagonizing the repressive H-NS-YmoA histone-like protein complex

The type III secretion system (T3SS) is a appendage used by many bacterial pathogens, such as pathogenic Yersinia, to subvert host defenses. However, because the T3SS is energetically costly and immunogenic, it must be tightly regulated in response to environmental cues to enable survival in the host. Here we show that expression of the Yersinia Ysc T3SS master regulator, LcrF, is orchestrated by the opposing activities of the repressive YmoA/H-NS histone-like protein complex and induction by the iron and oxygen-regulated IscR transcription factor. Although IscR has been shown to bind the lcrF promoter and is required for in vivo expression of lcrF, in this study we show IscR alone fails to enhance lcrF transcription in vitro. Rather, we find that in a ymoA mutant, IscR is no longer required for LcrF expression or T3SS activity. Additionally, a mutation in YmoA that prevents H-NS binding (ymoAD43N) rescues the T3SS defect of a {Delta}iscR mutant, suggesting that a YmoA/H-NS complex is needed for this repressive activity. Furthermore, chromatin immunoprecipitation analysis revealed that H-NS is enriched at the lcrF promoter at environmental temperatures, while IscR is enriched at this promoter at mammalian body temperature under aerobic conditions. Importantly, CRISPRi knockdown of H-NS leads to increased lcrF transcription. Collectively, our data suggest that as IscR levels rise with iron limitation and oxidative stress, conditions Yersinia experiences during extraintestinal infection, IscR antagonizes YmoA/H-NS-mediated repression of lcrF transcription to drive T3SS activity and manipulate host defense mechanisms. Author SummaryFacultative pathogens must silence virulence gene expression during growth in the environment, while retaining the ability to upregulate these genes upon infection of a host. H-NS is an architectural DNA binding protein proposed to silence horizontally acquired genes, regulating virulence genes in a number of pathogens. Indeed, H-NS was predicted to regulate plasmid-encoded virulence genes in pathogenic Yersinia. However, Yersinia H-NS is reported to be essential, complicating testing of this model. We used chromatin immunoprecipitation and inducible CRISPRi knockdown to show that H-NS binds to the promoter of a critical plasmid-encoded virulence gene, silencing its expression. Importantly, under conditions that mimic Yersinia infection of a mammalian host, the transcriptional regulator IscR displaces H-NS to drive virulence factor expression.

microbiology↗

bioRxiv2021

Profiling RT-LAMP tolerance of sequence variation for SARS-CoV-2 RNA detection

The ongoing SARS-CoV-2 pandemic has necessitated a dramatic increase in our ability to conduct molecular diagnostic tests, as accurate detection of the virus is critical in preventing its spread. However, SARS-CoV-2 variants continue to emerge, with each new variant potentially affecting widely-used nucleic acid amplification diagnostic tests. RT-LAMP has emerged as a quick, inexpensive diagnostic alternative to RT-qPCR, but has not been studied as thoroughly. Here we interrogate the effect of SARS-CoV-2 sequence mutations on RT-LAMP amplification, creating 572 single point mutation "variants" covering every position of the LAMP primers in 3 SARS-CoV-2 assays and analyzing their effects with over 4,500 RT-LAMP reactions. Remarkably, we observed only minimal effects on amplification speed and no effect on detection sensitivity, highlighting RT-LAMP as an extremely robust technique for viral RNA detection. Additionally, we describe the use of molecular beacons to sensitively identify variant RNA sequences. Together these data add to the growing body of knowledge on the utility of RT-LAMP and increase its potential to further our ability to conduct molecular diagnostic tests outside of the traditional clinical laboratory environment.

molecular biology↗

bioRxiv2021

Improving RT-LAMP Detection of SARS-CoV-2 RNA through Primer Set Selection and Combination

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has emerged as a viable molecular diagnostic method to expand the breadth and reach of nucleic acid testing, particularly for SARS-CoV-2 detection and surveillance. While rapidly growing in prominence, RT-LAMP remains a relatively new method compared to the standard RT-qPCR, and contribution to our body of knowledge on designing LAMP primer sets and assays can have significant impact on its utility and adoption. Here we evaluate 18 LAMP primer sets for SARS-CoV-2, comparing speed and sensitivity with different LAMP formulations and conditions across more than 5,000 RT-LAMP reactions and identifying several primer sets with similar high sensitivity for different SARS-CoV-2 gene targets. Significantly we observe a consistent sensitivity enhancement by combining primer sets for different targets, confirming and building on earlier work to create a simple, general approach to building better and more sensitive RT-LAMP assays.

molecular biology↗

bioRxiv2017

Inferential Learning of Serial Order of Perceptual Categories by Rhesus Monkeys (Macaca mulatta)

Category learning in animals is typically trained explicitly, in most instances by varying the exemplars of a single category in a matching-to-sample task. Here, we show that rhesus macaques can learn categories by a transitive inference paradigm in which novel exemplars of five categories were presented throughout each training session. Instead of requiring decisions about a constant set of repetitively presented stimuli, we studied the macaques ability to determine the relative order of multiple exemplars of particular stimuli that were rarely repeated. Ordinal decisions generalized both to novel stimuli and, as a consequence, to novel pairings. Thus, we showed that rhesus monkeys could learn to categorize on the basis of implied ordinal position, and that they could then make inferences about category order. Our results challenge the plausibility of association models of category learning and broaden the scope of the transitive inference paradigm.\n\nSignificance StatementThe cognitive abilities of non-human animals are of enduring interest to scientists and the general public because they blur the dividing line between human and non-human intelligence. Categorization and sequence learning are highly abstract cognitive abilities each in their own right. This study is the first to provide evidence that visual categories can be ordered serially by macaque monkeys using a behavioral paradigm that provides no explicit feedback about category or serial order. These results strongly challenge accounts of learning based on stimulus-outcome associations.

animal behavior and cognition↗