Jones, M. N. | Search bioRxiv

bioRxiv2023

LiaX is a surrogate marker for cell-envelope stress and daptomycin non-susceptibility in Enterococcus faecium

Daptomycin (DAP) is often used as a first line therapy to treat vancomycin-resistant Enterococcus faecium (VREfm) infections but emergence of DAP non-susceptibility threatens the effectiveness of this antibiotic. Moreover, current methods to determine DAP MICs have poor reproducibility and accuracy. In enterococci, DAP resistance is mediated by the LiaFSR cell membrane stress response system and deletion of liaR encoding the response regulator results in hypersusceptibility to DAP and antimicrobial peptides. The main genes regulated by LiaR are a cluster of three genes, designated liaXYZ. In Enterococcus faecalis, LiaX is surface exposed with a C-terminus that functions as a negative regulator of cell membrane remodeling and an N-terminal domain that is released to the extracellular medium where it binds DAP. Thus, in E. faecalis, LiaX functions as a sentinel molecule recognizing DAP and controlling the cell membrane response, but less is known about LiaX in E. faecium. Here, we found that liaX is essential in E. faecium (Efm) with an activated LiaFSR system. Unlike E. faecalis, Efm LiaX is not detected in the extracellular milieu and does not appear to alter phospholipid architecture. We further postulated that LiaX could be used as a surrogate marker for cell envelope activation and non-susceptibility to DAP. For this purpose, we developed and optimized a LiaX ELISA. We then assessed 86 clinical E. faecium BSI isolates for DAP MICs and used whole genome sequencing to assess for substitutions in LiaX. All DAP-R clinical strains of E. faecium exhibited elevated LiaX levels. Strikingly, 73% of DAP-S isolates by standard MIC determination had elevated LiaX ELISAs above the established cut-off. Phylogenetic analyses of predicted amino acid substitutions showed 12 different variants of LiaX without a specific association with DAP MIC or LiaX ELISA values. Our findings also suggest that many Efm isolates that test DAP susceptible by standard MIC determination are likely to have an activated cell stress response that may predispose to DAP failure. As LiaX appears to be essential for the cell envelope response to DAP, its detection could prove useful to improve the accuracy of susceptibility testing by anticipating therapeutic failure.

microbiology↗

bioRxiv2023

Dying of thirst: Osmoregulation by a hawkmoth pollinator in response to variability in ambient humidity and nectar availability

Climate-induced alterations in flowering phenology can lead to a temporal mismatch between pollinators and the availability of floral resources. Such asynchrony may be especially impactful in desert ecosystems, where flowering time and pollinator emergence are particularly sensitive to rainfall. To investigate the osmoregulation of a desert-living hawkmoth pollinator Manduca sexta, we sampled hemolymph osmolality of over 1000 lab-grown moths held at 20%, 50%, and 80% ambient humidity. Under starvation, the hemolymph osmolality of moths remained within a healthy range from days 1-3, regardless of ambient humidity. However, osmolality levels increase steeply from a baseline of 360-370 mmol/kg to 550 mmol/kg after 4-5 days in low and intermediate humidity and after 5 days in high humidity. Starved moths exposed to low humidity conditions died within 5 days, whereas those in high humidity conditions lived twice as long. Moths fed either synthetic Datura wrightii nectar, synthetic Agave palmeri nectar, or water, maintained osmolality within a healthy baseline range of 350-400 mmol/kg. The same was true for moths that fed on authentic floral nectars. However, moths consumed higher amounts of synthetic nectar, likely due to the non-sugar nectar constituents in the authentic nectar. Finally, simulating a 4-day mismatch between pollinator emergence and nectar availability, we found that a single nectar meal can osmotically rescue moths in dry ambient conditions. Our findings indicate that hemolymph osmolality provides a rapid and accurate biomarker for assessing both the health and relative hydration state of insect pollinators.

physiology↗