Biology subjects

Sheppard, S.

Publications and source records attributed to Sheppard, S..

3 recordsLinked to original sources

bioRxiv2018

Understanding the killing mechanism of action by virus-infected yeasts

Killer yeasts are microorganisms, which can produce and secrete proteinaceous toxins, a characteristic gained via infection by a virus. These toxins are able to kill sensitive cells of the same or a related species. From a biotechnological perspective, killer yeasts have been considered as beneficial due to their antifungal/antimicrobial activity, but also regarded as problematic for large-scale fermentation processes, whereby those yeasts would kill species off starter cultures and lead to stuck fermentations. Here, we propose a mechanistic model of the toxin-binding kinetics pertaining to the killer population coupled with the toxin-induced death kinetics of the sensitive population to study toxic action in silico. Our deterministic model explains how killer Saccharomyces cerevisiae cells distress and consequently kill the sensitive members of the species, accounting for the K1, K2 and K28 toxin mode of action at high or low concentrations. The dynamic model captured the transient toxic activity starting from the introduction of killer cells into the culture at the time of inoculation through to induced cell death, and allowed us to gain novel insight on these mechanisms. The kinetics of K1/K2 activity via its primary pathway of toxicity was 5.5 times faster than its activity at low concentration inducing the apoptotic pathway in sensitive cells. Conversely, we showed that the primary pathway for K28 was approximately 3 times slower than its equivalent apoptotic pathway, indicating the particular relevance of K28 in biotechnological applications where the toxin concentration is rarely above those limits to trigger the primary pathway of killer activity.

systems biology↗

bioRxiv2017

Failure of in vitro differentiation of Plasmodium falciparum gametocytes into ookinetes arises because of poor gamete fertilisation

A critical step towards malaria elimination will be the interruption of Plasmodium transmission from the human host to the mosquito. At the core of the transmission cycle lies Plasmodium sexual reproduction leading to zygote formation and mosquito midgut colonisation by ookinetes. Whilst in vitro ookinete culture from the murine and avian malaria parasites, Plasmodium berghei and P. gallinaceum, has greatly increased our knowledge of transmission biology; efforts to mimic the process in the human parasite P. falciparum have, to date, had only limited success. Using fluorescence microscopy and flow cytometry with antibodies specific to the male gametocyte and developing ookinetes, we sought to evaluate P. falciparum ookinete production using previously published in vitro protocols. We then compared in vitro versus in vivo ookinete production in both P. falciparum and P. berghei parasites, exploring potential barriers to complete development. Finally, we sought to test a wide range of literature-led culture conditions towards further optimisation of in vitro P. falciparum ookinete production. Despite extensive testing, our efforts to replicate published methods did not produce appreciable quantities of fully formed P. falciparum ookinetes in vitro. In parallel, however, gametocyte cultures that failed to differentiate fully in vitro successfully developed into ookinetes in vivo with an efficiency approximating that of P. berghei. Flow cytometry analysis showed that this disparity likely lies with the poor fertilization of P. falciparum gametes in vitro. Attempts to improve gametocyte fertility or define conditions more permissive to fertilisation/ookinete survival in vitro were also unsuccessful. Current in vitro conditions for P. falciparum ookinete production are not optimal for gamete fertilisation either due to the lack of parasite-species-specific mosquito factors missing from in vitro culture, or non-permissive cues contaminating culture preparations.

microbiology↗

bioRxiv2017

Epistasis mediated alleviation of the cost of antibiotic resistance for MRSA

Understanding how multi-drug resistant pathogens evolve is key to identifying means of curtailing their further emergence and dissemination. Fitness costs imposed on bacteria by resistance mechanisms are believed to hamper their dissemination in an antibiotic free environment, however, some have been reported to have little or no cost, which suggests there are few barriers preventing their global spread. One such apparently cost-free resistance mechanism acquired by the major human pathogen Staphylococcus aureus is to the clinically important antibiotic mupirocin, which is mediated by mutation of the highly-conserved and essential isoleucyl-tRNA synthethase (ileS) gene. In Genome Wide Association Studies (GWAS) on two genetically and geographically distinct MRSA lineages we have found this mutation to be associated with changes in bacterial virulence, driven through epistatic interactions with other loci. Given the potential dual effect of this mutation on both antibiotic resistance and virulence we adopted a proteomic approach and observed pleiotropic effects. This analysis revealed that the activity of the secretory apparatus of the PSM family of cytolytic toxins, the Pmt system, is affected in the mupirocin resistant mutant, which explains why it is less toxic. As an energetically costly activity, this reduction in toxicity masks the fitness costs associated with this resistance mutation, a cost that becomes apparent when toxin production is required. Given the widespread use of this antibiotic, and that this resistance often results from a single nucleotide substitution in the ileS gene, these hidden fitness costs provide an explanation for why this resistance mechanism is not more prevalent. This work also demonstrates how population-based genomic analysis of virulence and antibiotic resistance can contribute to uncovering hidden features of the biology of microbial pathogens.

microbiology↗